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Resting memory CD4+ T cells are the largest reservoir of persistent infection in HIV-1-positive subjects. They harbor dormant, stably integrated virus despite suppressive antiretroviral therapy, posing an obstacle to a cure. Surface markers that identify latently infected cells remain unknown. Microarray analyses comparing resting latently infected and uninfected CD4+ T cells generated in vitro showed profound differences in the expression of gene programs related to transcriptional and posttranscriptional regulation, cell proliferation, survival, cycle progression, and basic metabolism, suggesting that multiple biochemical and metabolic blocks contribute to preventing viral production in latently infected cells. We identified 33 transcripts encoding cell surface markers that are differentially expressed between latently infected and uninfected cells. Quantitative reverse transcriptase PCR (RT-QPCR) and flow cytometry analyses confirmed that the surface marker CD2 was expressed at higher levels on latently infected cells. To validate this result in vivo, we sorted resting memory CD4+ T cells expressing high and low surface levels of CD2 from six HIV-1-infected subjects successfully treated with antiretroviral drugs for at least 3 years. Resting memory CD4+ CD2high T cells from all subjects harbored higher HIV-1 DNA copy numbers than all other CD4+ T cell subsets. Moreover, after ex vivo viral reactivation, robust viral RNA production was detected only from resting memory CD4+ CD2high T cells but not from other cell subsets. Altogether, these results show that a high CD2 expression level is a hallmark of latently infected resting memory CD4+ T cells in vivo.
Current antiretroviral therapy (ART) is ineffective at eradicating human immunodeficiency virus type 1 (HIV-1) infection. Low-level viremia (<50 copies/ml) is often detected with ultrasensitive assays in patients on suppressive ART (1); occasional bursts of viral replication (“blips”) occur (2, 3), and eventually, viremia rebounds after ART interruption in all but exceptional cases (4).
HIV-1 establishes persistent infection in a number of cell types at different anatomical compartments via different mechanisms (reviewed in references 5, 6, 7, 8, and 9). Latent reservoirs harbor stably integrated HIV-1 DNA in the absence of viral particle production, are refractory to antiretroviral drugs, and are invisible to immune surveillance. The largest latent reservoir consists of resting memory CD4+ T cells, with a major contribution from central and transitional memory CD4+ T cells (10).
Numerous factors, such as the quiescent nature of resting memory CD4+ T cells, the site of viral integration, and many others, contribute to the establishment and maintenance of HIV-1 latency. Some of these mechanisms are currently being exploited to flush the latent provirus from its reservoirs in the absence of T cell activation (5, 11–13) by combining latency-reversing agents (LRAs) that reactivate latent proviruses, causing the demise of the infected cell, with highly active ART (HAART) intensification, which prevents infection of new target cells. While this approach presents potential pitfalls (the fraction of replication-competent latent proviruses that can be reactivated with LRAs [A. Cillo, M. Sobolewski, J. Coffin, and J. Mellors, presented at the 20th Conference on Retroviruses and Opportunistic Infections, Atlanta, GA, 2013; Y. C. Ho, L. Shan, J. Wang, N. Hosmane, J. Blankson, and R. Siliciano, presented at the 20th Conference on Retroviruses and Opportunistic Infections, Atlanta, GA, 2013], the fraction of latently infected CD4+ T cells that survives after viral reactivation , incomplete efficacy of HAART in sanctuary sites [15–17], and the impact of LRAs on reservoirs other than CD4+ T cells and on other latent viruses [18, 19]), reactivation of latent reservoirs merits further investigation. Nevertheless, there is also a need to develop alternative therapeutic approaches to eliminate or reduce the number of latently infected cells. The identification of cell surface biomarkers would provide an impetus in that direction. Indeed, uniquely or differentially expressed surface molecules could be used to isolate a partially purified subpopulation of latently infected cells for further characterization. Moreover, they could provide new therapeutic targets for the elimination of latently infected cells even in the absence of viral reactivation. To gain insight into the cellular mechanisms contributing to viral latency, and with the goal of identifying distinctive markers that distinguish latently infected CD4+ T cells, we have used an in vitro model developed in our laboratory (20) to study the expression profile of latently infected CD4+ T cells by microarray analysis. The results that we report in this study point to new mechanisms for the establishment and maintenance of latency in CD4+ T cells that could be exploited for exploring novel therapies aimed at targeting this reservoir. In addition, this survey identified a panel of genes encoding cell surface molecules that were differentially expressed in latently infected versus uninfected cells, which may have diagnostic as well as therapeutic implications. Among the markers identified in our study, CD2 was particularly interesting because of its know therapeutic applications (21–31). Sorting of resting memory CD4+ T cells expressing high levels of the CD2 receptor from HIV-1-infected subjects on suppressive ART allowed a significant enrichment of latently infected cells able to produce robust levels of viral particles following ex vivo reactivation. Therefore, the studies presented below demonstrate that high levels of CD2 expression identify latently infected resting memory CD4+ T cells in virally suppressed HIV-1-infected subjects.
All of the subjects provided their informed written consent to participate in the study. Peripheral blood mononuclear cells (PBMCs) of 4 HIV-1-negative donors (donors 3, 111, 112, and 113) were obtained with signed informed consent, after approval of the Institutional Review Board of the University of Maryland, Baltimore. PBMCs of 6 HIV-1-seropositive subjects (subjects ST045, ST101, ST102, ST104, ST109, and ST113) with undetectable viremia on suppressive ART for at least 3 years were obtained with signed informed consent and approved by the Institutional Review Board at Martin Memorial Health Systems (Stuart, FL).
The model for generation of latently infected CD4+ T cells in vitro was described previously (20), except for the modifications described in the supplemental material.
Procedures for sorting of in vitro-generated latently infected cells, total RNA isolation, and microarray analysis are described in the supplemental material and were described previously (32).
Total RNA was isolated as described above, and cDNA was generated by using the high-capacity RNA-to-cDNA kit (Applied Biosystems). Quantitative real-time PCRs (QPCRs) were performed in triplicate on a Bio-Rad IQ5 instrument by using TaqMan gene expression assays (Applied Biosystems) (see Table S4 in the supplemental material), according to the manufacturer's instructions. Expression levels were compared to the levels of MED19, since it did not show differential expression in the microarray.
Surface expression of CD2, CD6, and CD130 was analyzed on CD4+ T cell cultures latently infected with HIV-1 carrying a green fluorescent protein (GFP) reporter gene in place of Nef (pNL4-3-GFP). Further details are available in the supplemental material.
We obtained PBMCs from 5 HIV-1-seropositive subjects with undetectable viremia who were on suppressive ART for at least 3 years (see Table S3 in the supplemental material). CD4+ T cells were enriched from PBMCs by negative selection. Quantification of total HIV-1 DNA was performed with 300,000 cells of each subset by using ultrasensitive HIV-1 DNA PCR assays (sensitivity, 1 copy), as previously described (10). For viral reactivation, naive CD4+ T cells and resting memory CD4+ T cells expressing high or low levels of CD2 (5 × 105 cells per subset) were stimulated with anti-CD3/CD28 Dynabeads for 6 days in the presence of antiretrovirals (180 nM zidovudine [AZT], 100 nM efavirenz [EFV], and 200 nM raltegravir [RALT]) to prevent new rounds of infection. We then used ultrasensitive reverse transcriptase PCR (RT-PCR) (detection limit, 1 HIV-1 RNA copy) to quantify HIV-1 RNA copies in supernatants. Finally, the percentages of naive, central memory, transitional memory, effector memory, and terminally differentiated cells were determined by flow cytometry by gating on total CD4+ T cells and on the subset of CD4+ T cells expressing high and low levels of CD2. Further details for all these procedures are provided in the supplemental material.
Complete results from this comparative host gene expression profiling analysis can be found at the public Gene Expression Omnibus (GEO) database under accession number GSE40550.
Latently infected cells were generated in vitro from CD4+ T cells of 4 HIV-1-negative donors (donors 3, 111, 112, and 113) according to our previously described model (20), with the modifications shown in Fig. S1A in the supplemental material and described in Materials and Methods. After infection and expansion, cells were allowed to rest for 1 week, which allowed them to achieve cell quiescence, as shown by the lack of the activation markers HLA-DR, CD69, and Ki67 (see Fig. S1B in the supplemental material).
We have previously shown that the HIV-1 p24gag antigen synthesized during productive infection persists in the cytoplasm of infected cells for several days and slowly declines during the latency phase (20). Detection of p24gag in the cytoplasm of latently infected cells does not reflect new rounds of viral infection or de novo synthesis, in that the addition of AZT or cycloheximide does not affect the slope of p24gag decay (20). Moreover, RT activity was detectable in culture supernatants of cells from two different donors at the peak of infection, but it was undetectable after 1 week of rest, indicating that by that time, active viral production had ceased (see Fig. S1C in the supplemental material). Therefore, we exploited cytoplasmic p24gag to sort latently infected from uninfected cells from the same initial cell culture by flow cytometry, as we described recently (32). As cytoplasmic p24gag declines over time, the frequency of p24gag-negative latently infected cells sorting with, and thus contaminating, the truly HIV-1-negative population grows as a function of the length of the resting phase. We circumvented this potential pitfall by employing a resting phase of 1 week, since this was shown to be sufficient to obtain quiescent, latently infected cells (see above).
Total RNA was isolated from sorted latently infected and uninfected cells generated from CD4+ T cells of four different donors. Paired RNA samples from infected and uninfected cells were labeled with Cy3 and Cy5 to allow dual-color competitive hybridization (20). Moreover, to control for the dye bias in our experiments, we implemented a dye swap protocol (reciprocal labeling) for paired RNA samples from 2 donors, as described previously (20), in Materials and Methods, and in Fig. 1A. RNA quality diagnostics did not reveal any artifacts (20).
Complete results from this comparative host gene expression profiling analysis can be found in Table S1 in the supplemental material and at the public GEO database (accession number GSE40550). A total of 533 genes were upregulated in latently infected cells, while 342 genes were downregulated (false discovery rate [FDR], <5%). Of particular interest are a panel of genes downmodulated in latently infected cells that could contribute to halt HIV-1 transcription due to their involvement in cellular metabolism, chromosome organization, gene expression, cell activation, proliferation, survival, and cell cycle progression (Fig. 1B).
Some of the factors known to have a role in HIV-1 infection and latency were also deregulated in latently infected cells. Thus, the mRNA of the chromatin-remodeling enzyme responsible for transcription repression, histone deacetylase 1 (HDAC1) (33), and the natural antiviral factor apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (APOBEC3G) (34) were up- and downregulated, respectively, in latently infected compared to uninfected cells. We also found reduced expression levels of a number of transcripts that might potentially impact entry into or maintenance of HIV-1 latency: mRNAs for several eukaryotic initiation-of-translation factors (EIFs), 1AX, 2A, 4A1, 4B, 4G2, and 5A (required for Rev function ); CCND2 and CCND3 (involved in G1 progression and G1/S transition in human T cells ); and MYST4 (a histone methyltransferase involved in chromatin condensation ) were expressed at lower levels in latently infected than in uninfected cells.
Finally, a number of transcripts involved in various steps of the virus life cycle were also found to be differentially modulated when latently infected cells were compared to uninfected cells from the same culture. For instance, we found that the transcripts of the HIV-1-suppressive factors CCL4/MIP-1β and CCL5/RANTES were downregulated in latently infected cells. However, mRNAs of the HIV-1 receptor CD4 and coreceptor CXCR4 genes were upregulated in these cells. Transcripts of factors involved in RNA splicing (PRPF4B, SFRS16, and SFRS17A) were upregulated in latently infected cells, whereas factors mediating nuclear import of Rev (IPO5/RANBP5  and IPO7 ) and Tat (NUP93 and NUP153 ) were downmodulated in these cells.
The results of our microarray analysis also revealed deregulated expression of many genes in Gene Ontology (GO) categories such as nucleic acid, protein, and lipid metabolic processes; RNA processing; posttranscriptional regulation; posttranslational protein modifications; protein localization; and nucleocytoplasmic transport. Moreover, genes involved in vesicle transport, endocytosis, and membrane invagination were also differently regulated in latently infected cells. Data for additional enrichment analyses, including the updated analysis of functional annotation (AFA), can be found at the GEO database (accession number GSE40550).
In relation to proliferation or survival, both pro- and antiapoptotic factors were found to be up- and downmodulated in latently infected cells. Thus, based on these results, it is difficult to ascertain whether latent infection confers a cell survival benefit.
To validate our data set, we performed real-time RT-PCR with total RNA from latently infected and uninfected cells from 2 donors for 15 genes identified in the microarray analysis. The results obtained by quantitative RT-PCR (RT-QPCR) for all the genes were in accordance with the differential expression found by microarray analyses: 5 out of 5 genes were upregulated (Fig. 2A), and 10 out of 10 were downregulated (Fig. 2B).
We found that 89 transcripts for membrane-associated proteins were differentially expressed (56 up- and 33 downregulated) in latently infected compared to uninfected CD4+ T cells generated in vitro (see Table S2 in the supplemental material). Among these 89 transcripts, 33 encode cell surface markers belonging to the cluster-of-differentiation (CD) protocol, including 20 genes upregulated and 13 downregulated in latently infected CD4+ T cells (Table 1). We used RT-QPCR to validate differential expression of 10 upregulated and 3 downregulated transcripts with total RNA from cells generated with our in vitro latency model. As shown in Fig. 3A and andB,B, all 13 genes (except CD6 with RNA from donor 112) were validated in samples from two donors. Next, we decided to confirm whether some of these receptors were also deregulated at the protein level by flow cytometry. We focused on surface molecules that were upregulated, since they may have more therapeutic and diagnostic potential, and we selected three receptors expressed at high (CD2), intermediate (CD6), and low (CD130/IL6RB) levels on CD4+ T cells. We used our in vitro model to generate cultures latently infected with full-length, replication-competent HIV-1 expressing GFP, which facilitates flow cytometry analysis. As GFP, like HIV-1 p24gag, persists in the cytoplasm of infected cells for several days during the latency phase (see Fig. S2 in the supplemental material), we measured the expression levels of these three markers on latently infected and uninfected cells by gating on the GFP-positive (GFP+) and GFP-negative (GFP−) fractions, respectively. The results were validated by using latently infected cultures generated using our in vitro model of HIV-1 latency. As shown in Fig. 3C, surface expression levels of all three receptors were higher on latently infected than on uninfected cells.
Next, we sought to determine whether the cell surface markers identified through the microarray analysis described above could be used to identify latently infected cells in vivo. As the differential expression level of the CD2 receptor between latently infected and uninfected cells in vitro was higher than those of CD6 and CD130 (Fig. 3C), we chose to perform our studies with clinical samples using CD2 first. Thus, we obtained a large number of PBMCs (5 × 109 to 15 × 109 cells) through leukapheresis from six HIV-1-seropositive subjects with undetectable viremia and who were on suppressive ART for at least 3 years (see Table S3 in the supplemental material). We enriched CD4+ T cells by negative selection, and we stained these cells with fluorochrome-conjugated antibodies to CD3, CD4, HLA-DR, CD45RA, and CD2, as described in Materials and Methods. After gating on the live-cell population, we sorted naive CD4+ T cells (CD3+ CD4+ CD45RA+), activated memory CD4+ T cells (CD3+ CD4+ HLA-DR+ CD45RA−), and resting memory CD4+ T cells (CD3+ CD4+ HLA-DR− CD45RA−) that fell within the 20th highest and lowest percentiles of CD2 fluorescence (CD2high and CD2low, respectively) (Fig. 4A). First, we measured the copies of HIV-1 DNA in the 4 sorted cell subsets, using CD4+ T cells as controls. We found that resting memory CD4+ CD2high T cells from all six subjects harbored higher HIV-1 DNA copy numbers than the other cell subsets (Fig. 4B). The median of the enrichment of HIV-1 DNA copies in the CD4+ CD2high subset compared to total CD4+ T cells was 5.7-fold (range, 3- to 10.8-fold; P = 0.013) (Fig. 4B).
Next, we measured viral RNA production following in vitro reactivation of resting memory CD4+ CD2high, resting memory CD4+ CD2low, and naive CD3+ CD4+ CD45RA+ T cells. Sorted cells from 3 of the 6 subjects analyzed as described above (subjects ST101, ST104, and ST113) were stimulated with anti-CD3/CD28 Dynabeads for 6 days in the presence of antiretrovirals to prevent new rounds of infection. We then used ultrasensitive RT-PCR to quantify HIV-1 RNA copies in culture supernatants after pelleting of viral particles by ultracentrifugation. We detected robust levels of viral RNA production in the culture medium of resting memory CD4+ CD2high T cells, whereas little or no viral particles were detected in the supernatants of resting memory CD4+ CD2low and naive CD4+ T cells (Fig. 4C). Thus, these results indicate that a high expression level of the surface receptor CD2 identifies resting memory CD4+ T cells harboring replication-competent HIV-1 in peripheral blood of virally suppressed patients.
Finally, we used flow cytometry to determine the frequencies of all major cell differentiation subsets (naive, central memory, transitional memory, effector memory, and terminally differentiated) within the total CD4+ T cell, CD4+ CD2low, and CD4+ CD2high populations of 5 out of 6 subjects as described above. As shown in Fig. 5, the CD4+ CD2high population was markedly enriched in cells displaying a memory phenotype. Thus, these results are in line with a previous study showing that integrated HIV-1 is found primarily within central (CD45RA− CCR7+ CD27+), transitional (CD45RA− CCR7− CD27+), and effector (CD45RA− CCR7− CD27−) memory CD4+ T cells (10).
Several limitations hinder the study of HIV-1 latency with clinical samples, including the lack of distinctive surface markers for latently infected cells, the low frequency of latently infected resting memory CD4+ T cells in ART patients, and the fact that clinical samples allow studying only reactivation from latency but not the mechanisms underlying its establishment. These reasons have compelled several groups to develop in vitro models to generate and study proviral latency in resting CD4+ T cells (20, 41–50; reviewed in reference 9).
The model developed in our laboratory (20) recapitulates the activation of primary CD4+ T cells via T cell receptor (TCR) engagement, infection with HIV-1, and return back to quiescence through a resting phase in the presence of low levels of interleukin-7 (IL-7). Our model is amenable to providing new insights into the nature of latency and identifying new biomarkers and therapeutic targets. The modifications of our original model that we adopted in the current study for the purpose of isolating latently infected cells—most notably a shortened resting phase—did not affect our ability to generate truly quiescent latently infected cells, as shown by the lack of surface and intracellular activation markers (HLA-DR, CD69, and Ki67) and by the absence of RT activity in the culture supernatants. The choice of using RT activity as a measure of viral latency was dictated by the evidence that cell-associated viral RNA can be detected in latently infected resting memory CD4+ T cells of HIV-1-infected subjects under effective antiretroviral therapy (11, 51), and therefore, this parameter might not provide a stringent measure of latency. This result underscores the role of low doses of IL-7 in the establishment of immunological memory in CD4+ T cells (52–54). We have previously shown that the HIV-1 antigen p24gag produced during active viral replication persists in the cytoplasm of infected cells for several days, even during latent infection, which allows us to distinguish latently infected CD4+ T cells from their uninfected counterparts (20). Thus, we took advantage of this marker to perform, for the first time, comparative genomic profiling of primary latently infected resting memory CD4+ T cells versus their uninfected counterparts sorted from the same culture. Previous studies were carried out with latently infected cell lines (55–57), but no studies using primary CD4+ T cells have been reported. Krishnan et al. showed altered expression levels of genes encoding components of the proteasome, histone deacetylases, and transcription factors in the chronically infected cell lines ACH-2, J1.1, and U1 compared to the parental uninfected cell lines A3.01, Jurkat, and U937 (56). The same data set was used later on to identify several networks at the latent stage, including an enrichment of proteins associated with HIV-1 replication, apoptosis, and cell growth regulation (58). Since latently infected cells cannot be distinguished from uninfected cells in vivo, only a comparative study of resting memory CD4+ T cells from viremic and aviremic subjects has been done so far (59).
Our microarray analysis indicates that latently infected resting memory CD4+ T cells have a very different gene expression profile than uninfected cells. It should be noted that because of the temporal course of our experimental design, it is unclear whether the genes identified in our screen are related to entry into latency, maintenance of latency, or both. Resolving this issue will require a more accurate follow-up study. The affected gene ontology not only covers pathways related to transcriptional and posttranscriptional regulation, as described previously (13, 60), but also illustrates more profound changes affecting cell proliferation, cell cycle progression, as well as basic cellular functions such as nucleic acid, lipid, carbohydrate, and amino acid metabolic processes. Altogether, these changes may contribute to the establishment of a cellular environment that is conducive to cell quiescence and viral latency. Recent ex vivo studies suggested that a single treatment with current LRAs induces viral reactivation in only a small percentage of latently infected cells (Cillo et al., presented at the 20th Conference on Retroviruses and Opportunistic Infections; Ho et al., presented at the 20th Conference on Retroviruses and Opportunistic Infections). Furthermore, recent in vivo studies showed that administration of a single dose and multiple doses of the HDAC inhibitor vorinostat led to transient increases in acetylation of histone H3 and in HIV-1 gag cell-associated RNA copy numbers. Therefore, treatment with vorinostat also affects the transcription activity of the HIV-1 long terminal repeat (LTR) in vivo. However, this effect was not paralleled by a spike in viremia (11; J. Elliott, A. Solomon, F. Wightman, M. Smith, S. Palmer, M. Prince, J. Watson, J. Hoy, J. McMahon, and S. R. Lewin, presented at the 20th Conference on Retroviruses and Opportunistic Infections, Atlanta, GA, 2013). A possible interpretation of these results may be that an increase in the level of cell-associated viral RNA following exposure to LRAs did not lead to viral production by latently infected cells. The results of our microarray analysis may suggest that latently infected cells lack the proper biochemical machinery required to support viral production despite higher intracellular levels of viral RNA. The use of concomitant LRAs targeting different transcription blocks was clearly shown to be more effective than single-agent approaches in vitro (43, 61). Moreover, it is conceivable that targeting of more downstream steps, including some of the ones identified in this study, may lead to more sustained viral production, especially in more refractory latent reservoirs.
The establishment of HIV-1 latency has been portrayed largely as a passive process. In other words, HIV-1 expression is shut down—and consequently, latency is achieved—as part of the process by which the host cell returns to quiescence. However, it is plausible that viral proteins expressed during productive infection of activated CD4+ T cells could play an active role by deregulating gene programs within the host cell so as to generate a cellular environment conducive to cell quiescence and viral latency. Indeed, our microarray study indicated that in latently infected cells, gene programs were modulated in a way that is consistent with cell quiescence, cell survival, transcription silencing, and evasion of the immune system. Since HIV-1 proteins have a profound effect on many host cell functions that are relevant to its life cycle (55, 58, 62–69), it is conceivable that they might also affect cell functions relevant to latency. On the other hand, it is possible to speculate that a subset of CD4+ T cells with a gene expression profile similar to the one emerging from our microarray analysis already preexists in peripheral blood of healthy individuals, so that upon HIV-1 infection, it provides an ideal environment for the establishment of latency.
Our microarray analysis found that the expressions of transcripts encoding a number of cell surface markers were deregulated in latently infected CD4+ T cells. This finding is of particular relevance since specific surface biomarkers to distinguish these cells have not yet been identified. Of these, CD2 was selected for validation in clinical samples, using peripheral blood of aviremic HIV-1-infected subjects under suppressive antiretroviral therapy for at least 3 years. Selection based on high expression levels of this CD2 receptor allowed strong ex vivo enrichment of a bona fide latent reservoir of resting memory CD4+ T cells. In all subjects analyzed, this CD2high population was shown to contain higher copy numbers of HIV-1 DNA and to give rise to more robust viral production upon reactivation than total resting memory CD4+ T cells. On the other hand, our data also show that the majority of CD2high cells are not infected. Moreover, we detected HIV-1 sequences in the CD2low subset of 5/6 subjects as well as HIV-1 RNA in the culture supernatant of CD2low cells of 1/3 subjects following ex vivo reactivation. Therefore, expression of high CD2 levels does not fully capture the latent reservoir, and while it represents a useful tool for future research, it is a limited one. The identification of new markers remains a high priority, and the list of transcripts identified in our microarray study might represent a valid starting point.
Two lines of evidence provide a possible basis for the identification of CD2high as a biomarker of bona fide latently infected CD4+ T cells. Indeed, binding of CD2 to its ligand, CD58/LFA-3, facilitates T cell interactions with antigen-presenting cells and perhaps enhances virus transmission. Additionally, LFA-3 is found on the membrane of HIV-1 virions and has been proposed to mediate viral targeting to particular tissues and specific cell types (70). Thus, cells overexpressing CD2 might bind HIV-1 and become infected more readily.
CD2 might represent a potential target for the elimination of latently infected CD4+ T cells. Whereas some anti-CD2 antibodies induce T cell proliferation (71) and viral transcription in PBMCs and resting CD4+ T cells (72, 73), binding of certain epitopes on CD2 induces T cell death via antibody-dependent cellular cytotoxicity (ADCC) induced by NK cells and activated monocytes. For instance, alefacept, a fusion protein composed of the first extracellular domain of human LFA-3 fused to the hinge, CH2, and CH3 domains of human IgG1, as well as rat IgG2b monoclonal antibody LO-CD2a/BTI-322 inhibit T cell proliferation and induce NK cell-mediated death of T cell memory subsets (74, 75). LO-CD2a/BTI-322 and its humanized version (siplizumab; MEDI-507) have been used for the treatment of acute kidney transplant rejection (22, 26, 28, 30), acute graft-versus-host disease (GVHD) (21, 24, 29), and psoriasis (23) and is being tested for the treatment of T cell malignancies (25, 27, 31). In addition, alefacept is FDA approved to kill memory T cells expressing higher levels of CD2 for the treatment of chronic psoriasis (76, 77). These studies suggest the possibility of targeting CD2 for the depletion of latently infected CD4+ T cells in HIV-1 infection. Although CD2 is also expressed on uninfected T and NK cells, the FDA has approved targeting of receptors such as CD20 for cell depletion in autoimmune diseases and cancer, despite the fact that CD20 is also expressed on normal B and T cells (78, 79).
Besides CD2, our microarray study found other interesting receptors that, on the basis of their known function and signaling pathways, could be explored to target or deplete this latent reservoir (Table 1). For example, engagement of the tumor necrosis factor (TNF) receptors CD262, CD270, and APO-3, found upregulated in our microarray in latently infected resting CD4+ T cells, triggers caspase activation and apoptosis (80, 81). In addition, two receptors listed in Table 1 are known to trigger cell death through the p38 mitogen-activated protein kinase (MAPK) pathway. Thus, antibodies to CD70, a TNF family member (80, 82), trigger death of malignant cells (83). Also, it is well documented that CXCR4 mediates p38 MAPK-dependent cell death in HIV-1 infection (84, 85). In addition, we found differential expressions of transcripts encoding cell surface activation markers such as CD38, CD69, and CD154/CD40L. This result is intriguing because our flow cytometry analysis showed that CD69 is not expressed on the cell surface after 1 week of a resting phase, and it further underscores the need to validate microarray results at the protein level.
In summary, microarray analysis using HIV-1 latently infected resting memory CD4+ T cells generated with our in vitro model points toward possible new pathways of latency beyond transcription that could be explored as new therapeutic targets aimed at eradicating this reservoir. In addition, we identified a panel of surface makers differentially expressed in latently infected cells, which seem worth investigating for their potential use as biomarkers. Indeed, they might allow the enrichment of this latent reservoir for in-depth molecular studies, for monitoring the size of the latent reservoir in the clinical setting, as well as for the development of novel therapeutic strategies aimed at eradicating HIV-1. Among them, CD2 has proven to allow efficient enrichment of latently infected resting memory CD4+ T cells ex vivo.
This work was supported by National Institutes of Health grant AI084711 (F.R.). L.M. was also supported by NIH-NCI grant P30CA006973.
Published ahead of print 12 June 2013
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JVI.01297-13.


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Quebec’s 5,000 Wind Energy Workers Deserve Answers
Political and economic leaders appeal to Pauline Marois
QUÉBEC, April 23, 2013 – The uncertainty cannot continue for Quebec’s 5,000 wind energy workers. That was the message sent to Premier Pauline Marois by the many political and economic leaders who came to Québec from several of the province’s regions to demand that the government quickly solicit bids for at least 700 MW of wind energy, which would allow the wind energy sector to continue developing past 2015. The sector benefits the entire province, and in particular the communities that host and participate in wind energy projects.
“In recent months, the wind energy sector decided to adopt a constructive attitude, to talk, collaborate and partner with the government. Despite this approach, the government is silent, uncertainty persists and rumours abound, even though the wind energy sector injects approximately half a billion dollars into Quebec’s economy every year. Quebeckers can only wonder whether the government has the will to continue developing the Quebec wind sector and safeguard its thousands of jobs” said Jean-François Nolet, Vice-President of Policy and Government Affairs at the Canadian Wind Energy Association (CanWEA).
It must be remembered that in its Energy Strategy, the Quebec government committed to the production of 4,000 MW of wind energy between 2006 and 2015. On this basis, the industry developed, foreign companies settled in Quebec, educational institutions created new programs and new businesses came into being. “Not seeing this commitment through would be an unacceptable step backwards, as much for the workers and the sector as for the regions that have turned to wind energy to build their industrial base. Most importantly, wind energy provides Quebec with $87.6 million in fiscal and parafiscal revenues,” adds Jean-François Samray, President and CEO of the AQPER (Association québécoise de la production d’énergie renouvelable).
“Madam Premier, you cannot keep Quebec’s 5,000 workers—1,200 of whom are from the Gaspésie—in the dark,” declared New Richmond mayor Nicole Appleby on behalf of several municipalities, RCMs and the Union of Quebec Municipalities, which have agreed to demand a new call for tenders. “The Gaspésie is proud to contribute to Quebec’s economic development through wind energy, because the growth of the sector here has resulted in benefits that reach as far as Montréal.”
“The wind energy sector has had a positive impact on the entire province, with the jobs created in wind energy plants and company headquarters. Municipalities and RCMs have also benefited from multiple partnerships that have brought in additional opportunities for development,” said Maurice Bernier, Prefect of the Granit RCM.
“Local businesses like our own joined the sector in 2002 because its future was promising. Foreign companies have come to set up their Canadian head offices right here in Quebec. The expertise being developed here will be applied across North America,” stated Éric Bellemare, Director of Business Development at Marmen.
“Quebec was the ideal place for us to establish our North American headquarters, given its solid planning and long-term vision for the market,” explained Marc-Antoine Renaud, Quebec Manager at ENERCON Canada. “In this transition toward the next energy policy, the next 700 MW is needed to ensure the sector’s future.”
“Maintaining and developing wind energy is not just about the industry and its workers. Local communities and Quebec as a whole are also affected. Quebec is a North American leader in wind energy, and Montréal is the wind energy hub of eastern North America,” Nolet remarked.
Advocates of the socio-economic benefits of wind energy, who have come from all over Quebec, will head to the National Assembly at 2 p.m. to request a meeting with Pauline Marois. They will ask the Premier to end the uncertainty facing the industry’s 5,000 workers and their families. The wait has lasted long enough. If Premier Marois wants to keep Quebec’s wind energy sector healthy, she will have to finally solicit bids for the 700 MW of wind energy everyone has so eagerly awaited.
CanWEA is the voice of Canada’s wind energy industry, actively promoting the responsible and sustainable growth of wind energy on behalf of its almost 400 members. A national non-profit association, CanWEA serves as Canada’s leading source of credible information about wind energy and its social, economic and environmental benefits. To join other global leaders in the wind energy industry, CanWEA believes Canada can and must reach its target of producing 20 per cent or more of the country’s electricity from wind by 2025. The document WindVision 2025: Powering Canada's Future is available at www.canwea.ca.
About the AQPER
The mission of the Association québécoise de la production d’énergie renouvelable (AQPER) is to promote renewable energy among the general public, businesses and governments and to advocate for the responsible development of Quebec’s energy resources in all renewable sectors. The Association represents the interests of independent electricity producers concerned with the principles of sustainable energy.
For information and interviews

domain: news


text:
SALE!!! Tribal Feathers Matt Satin Georgette
Tribal Feathers Matt Satin Georgette
This is a light-weight matt satin georgette with a small amount of spandex. As it's very soft to touch, it's suitable for summer dresses, skirts and blouses. It can also be used as lining for coats and jackets, or you could simply make yourself a pair of luxurious-feeling pyjamas. Imagine a simple pair of baggy hareem pants or a simple halter slip dress for the beach.
Composition: 97% Polyester / 3% Spandex
Price is for 1 metre (after one metre, fabric comes in one complete length)

domain: design


text:
A Faerie Lair and Why Life is Like an Easter Egg Hunt
“Faerie Lair” by Rayna
(custom prints available)
I offer to you, this thought and visual image to meditate upon today…
There are small and hidden places that will always remain mysterious to the dreaming human being. The secrets and treasures these chambers must hold—but wait, no, they are in Truth empty spaces. The journey to find all of the answers is an unending quest for the weary thought addict. But if we knew every little detail, all would in fact become unknown! This is why life is like an Easter egg hunt: You are to discover that there is only One Truth. And You are IT.
Love and Light,

domain: artistic


text:
COLUMBUS, Ohio (AP) – A proposal targeting drug dealers in Ohio who traffic in products laced with the opioid fentanyl has cleared the state Senate.
The bill defines microscopic amounts of the dangerous painkiller, when not contained in authorized pharmaceutical form, as enough to trigger a drug offense under Ohio law.
The legislation cleared the chamber 25-2 in Thursday. It heads next to the House.
Under the proposal, providing someone with five milligrams or more of fentanyl or a gram or more of any substance containing fentanyl would constitute dealing in bulk under Ohio drug laws.
The measure comes as heroin, pills and marijuana laced with fentanyl are contributing to an epidemic of drug-related overdoses and deaths across Ohio and the U.S.


text:
Lessons in responsibility during a bad news week
Last week several stories led Desert Sage to ponder the difference between responsibility and guilt.
When you study ethics you learn about two kinds of responsibility. There is something called “role responsibility” that is often confused with “moral responsibility.” They are different. Imagine a first responder who says after a brave rescue: “I am not a hero, I was just doing my job.” In that humble statement, she makes a distinction between fulfilling a role (as a rescuer) and getting moral credit for it. Credit and blame have to do with moral responsibility. Whether or not you believe ethics has to do with a Creator, this is about culture. You own personal responsibility for your actions, yet you are not alone. As Willard Gaylin put it: “Freedom demands responsibility; autonomy demands culpability.” You exist as an individual and as part of a society.
For instance, there is the disturbing story of Kenneth Jehle, the Albuquerque schoolteacher accused of harassing and inappropriately touching numerous girls at three different schools starting in 2002. Despite multiple complaints by students, parents, coworkers, and even a police report filed by an officer of the Albuquerque Public Schools, Jehle’s career flourished until he was fired in 2014. His undoing only came after one family sued the district, Jehle himself, and two principals who allegedly covered up for him. Last week, APS settled the lawsuit for $750,000.
Jehle’s individual guilt is one matter, but a deeper question concerns how the district failed in its role, which is to maintain a safe learning environment. How did this problem continue for so long without Jehle being held accountable? Does APS not track teachers with a history of complaints? Why did Principal Sam Obenshain repeatedly dismiss complaints about this teacher, even bringing Jehle with him after he transferred to another school? Obenshain arguably put students in harm’s way by this negligence, yet he is still the head of that school —still charged with the safety and welfare of children. Unless there are visible and binding changes at APS, a key social obligation has been shirked despite the cash settlement.
We can be generous or stingy with our sense of communal responsibility. The men who shot up Paris on Nov. 13 may have shouted “Allahu akbar,” but they are not true representations of typical Muslims. Ordinary Muslims are not to blame for what they did. Nonetheless, Muslims around the world have spoken out to denounce terrorism as anti-Islamic, even as assaults on anyone construed to be a Muslim have risen drastically. This is a generous (but also defensive) expansion of a shared “role responsibility.”
Then came the shooting at a Planned Parenthood clinic in Colorado Springs on Nov. 27. Clinics providing abortions have been targets of domestic terrorism for decades. Operation Rescue and other anti-abortion groups have often encouraged tactics of harassment and fear, while distancing themselves from any responsibility for actual violence. Politicians play this game, too. Early indications suggest Robert Dear, the alleged shooter, may be deranged and not representative of a movement. Nonetheless, a pro-life activist in Las Cruces, Mark Cavaliere, generously expanded his “role responsibility” as a leader of Las Cruces for Life. He drew an ethical line where certain presidential candidates will not.

domain: sociology


Protocols of MSn Spectra Acquisition
These instructions are based on the condition that uses of a MALDI-QIT-TOF mass spectrometer . Please follow the CID spectra acquisition protocol below when you compare your spectra with the spectral image in GMDB.
Instrument: MALDI-QIT-TOF MS
CID spectra acquisition:
1. Place 0.5 μL of a sample solution on a target plate and allow it to dry. Cover the dried analyte on the target plate with 0.5 μL of the matrix solution (10 mg/mL of 2,5-dihydroxybenzoic acid in 30% ethanol) and allow it to dry. Then, recrystallize the dried material by adding 0.15 μL of 99.5% ethanol to the matrix-analyte mixture on the target plate.
2. Acquire MS spectra of glycans in the positive mode.
3. Acquire MS2 spectra of sodium adduct ions derived from the glycans. For the acquisition of CID spectra, adjust the CID energy so that the signal of precursor ion almost disappears. If the intensity of precursor ion in the spectrum is more than 15% of the base peak, the spectrum must be discarded and reacquired with a larger CID energy. Use an automatic acquisition function with the regular raster that governs the laser shot patterns.
4. Acquire MS3 spectra of all the major fragment ions in the MS2 spectra in the same manner as described above for MS2 acquisition.


The Mossberg 500 is uniquely issued to the US ArmyAssault kit on certain maps. It features a 6 round capacity. It has high power, a low rate of fire, low accuracy and a short range. Performance wise, it is the ultimate close quarters weapon in the game. Usually, it can effectively kill targets from 20 meters away with only 2 or 3 shots. In anywhere within 10 meters, it is highly capable of killing in a single shot, and its low accuracy can still somewhat help with hip fire. Its main disadvantage is its low rate of fire due to the pump-action function, and the lack of range.
Because Battlefield Vietnam features different loadouts for each faction, this list may not be specific enough. Please see each faction's respective page for information on individual weapon kits and loadouts.


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What is there to say about a place you’ve never been and do not know? A somewhere stumbled into by half accident?
It shouldn’t be a surprise to hear a woodpecker in a wood. It depends on the circumstances I suppose. In the last week of January, I was in a small wood on the edge of a housing estate in Lewisham – Hillcrest Wood. The sound of a great spotted woodpecker drumming isn’t yet uncommon, even in London, but here, as you will see, it felt unexpected, though enormously welcome.
Here were ancient trees, darkling trees, summer and winter trees, ancient oaks, looming pines, explosive cherries, laugh out loud at the wonder of it all trees. In one case a massive old volume was open on a page showing a 19th century photograph of Beech tree. Especially fascinating was the tree’s position on the side of a sunken lane, which meant that its multiple tangled roots were exposed to the world, in a glorious, twisting, serpentine display.
…there on the edge of the flat stretching roof
Stood a magpie, a gull and a pigeon
Neatly spaced –
The cast from some terrible joke.
A mysterious letter. A secret journal. An ancient wood, in borderland territory. Deceptive paths and strange, ghostlike figures, stirring at the edge of the trees. These were some of the elements that quickly took hold and drew me into Ryhope Wood, when I first read about it aged 15.
Recently I re-read it, rediscovered and found many new things to treasure.
Dead rats aside, there was one area of the garden that always made me feel a little uneasy. As the name suggests, there was once a mill here.
In the late 18th century it was a paper mill, but by the 1820s had become a cloth-mill. By the end of the Victorian era, the mill was gone, but perhaps something else lingered in the grounds.
I’ve always quite liked the style of old handbills: the erratic punctuation, jumbled type sizes and overuse of exclamation marks especially. If I produced a handbill for this blog, it would look something like this…

domain: geography


What do you get if you mix a Legendary and Iconic Hip Hop duo from NYC with a Legendary UK Hip Hop group ???…..Infamous Murder Mix!
The results connect and make perfect Hip Hop sense. The accents , vocal tones and slang editorials are different but the energy , vividness of speech and street connectivity are real.
Salutes to our Homie The UK award winning Radio Producer/DJ Asante for letting us know about this gem of audio dopeness. This mix fuses the UK’s Terra Firma featuring Richochet Klashnekoff, Skriblah and Kyza Smirnoff with Prodigy & Havoc of The Legendary Mobb Deep. Enjoy!


text:
Eppley Limited (EPLY) has advised that the Company has effectively refinanced its US$ Preference Shares.
In a stock exchange notification, the company said, “As a result of requisite approvals received from holders of the US$ Preference Shares, the maturity date with respect to the US$ Preference Shares is now January 24, 2024 and the agreed dividend rate will be 6 per cent per annum, payable monthly, effective January 23, 2021.
Eppley’s Managing Director Nicholas Scott said the preference shares originally had a dividend rate of 5 per cent with a redemption date of January 23, 2021.
Following the transaction, the dividend rate remains five per cent until January 23, 2021 and thereafter will be six per cent until the new redemption date in 2024.
” This reflects the longer duration of the preference shares and market conditions.”– Eppley’s Managing Director Nicholas Scott
“The transaction demonstrates the capital market’s continued confidence in Eppley as a result of its long track record of consistently strong returns. It also improves Eppley’s mature profile, boosts its liquidity and allows it to take advantage of investment opportunities in its pipeline.”
Eppley provides loans and insurance premium financing.
The company produced earnings per share of $0.21 in the first quarter of 2020, a 6.5 per cent return for shareholders1
At the end of the quarter, net asset value was $4.08 per share. Proprietary Investment Portfolio at the end of the quarter we owned a $3.9 billion investment portfolio consisting of loans, leases, receivables and investments in mezzanine, real estate, infrastructure and asset management joint ventures and subsidiaries. The average income yield of our portfolio was 12 per cent.
The company cites portfolio diversification as its principal strength, with investments across multiple asset classes “many of which like real estate, infrastructure and secured loans and leases are defensive.” The company also indicated that it is not heavily exposed to any one industry, including tourism.
Directors added, “Our asset management business provides a stable stream of recurring earnings largely insulated from credit, market or liquidity risks.
The company says that focus in 2020 will be on navigating the landscape created by COVID 19 “and preparing to seize opportunities as they emerge.”
Eppley reported at year end 2019 that it has produced compounded average annual returns to shareholders of over 35 per cent as a public company.
At year-end 2019, its investment portfolio was $3.8 billion and the average return on portfolio was 12 per cent.


text:
Ottawa, September 1, 2011 - The Canadian Food Inspection Agency (CFIA) and Johnvince Foods are warning the public not to consume certain prepackaged raw shelled walnut products described below because these products may be contaminated with E. coli O157:H7.
The following raw shelled walnut products, imported from USA and packaged in Canada,
are affected by this alert.
|Brand & Product||Size||UPC||Code|
Raw California Walnut Halves Unsalted
|250 g||0 60383 87185 7||Best Before 2012 OC 07|
|Reddi Snack Hand Selected
|350 g||0 64777 28695 1||16581|
There have been no confirmed illnesses associated with the consumption of these products.
Johnvince Foods, Downsview, ON is voluntarily recalling the affected products from the marketplace. The CFIA is monitoring the effectiveness of the recall.

domain: food


text:
I just love you Sven! You are such a romantic and a true gentleman. The lady in your life is so lucky.
~~~~~~~~~~~~~~~~~~~~~~~~~ I hope you still feel small when you stand beside the ocean. Whenever one door closes I hope one more opens. Promise me you'll give faith a fighting chance. And when you get the choice to sit it out or dance - I hope you dance. song by LeeAnn Womack ~~~~~~~~~~~~~~~~~~~~~~~~~
Florida's Foreverly Shores

domain: literature


text:
"I want you to know, as you read me, precisely who I am and where I am and what is on my mind. I want you to understand exactly what you are getting; you are getting a woman who for some time now has felt radically seperated from most of the ideas that seem to interest other people. You are getting a woman who somewhere along the line misplaced whatever slight faith she ever had in the social contract, in the meliorative principle, in the whole grand pattern of human endeavor."
– Joan Didion, The White Album
A glowing article in the New York Review of Books about Joan Didion set me off on a spree of reading her early work – which has turned out to be the high point of my reading for the month. In the article, John Leonard writes: "I've been trying for four decades to figure out why her sentences are better than mine or yours…something about cadence. They come at you, if not from ambush, then in gnomic haikus, icepick laser beams, or waves." I couldn't agree more. Didion is one of those rare writers whose style feels so balanced, so clean, that you enjoy reading them even when they write about virtually nothing (remember Hemingway in A Moveable Feast?). There's something about her writing that escapes definition but is undeniable – her lines have a gravity all their own.
The White Album is a brilliant example of this. The title piece, with its flawless evocation of the paranoia and restlessness of the 60's (complete with college protests, Black Panthers and a dream-like evening with the Doors), is exciting enough, but the really stand-out piece for me was an essay on the feminist movement. Didion starts by celebrating the feminist movement as the ultimate class struggle, but soon falls to critiquing it on grounds that I've always strongly agreed with . The problem with the way the feminist movement has evolved, Didion suggests, is that it has placed the Everywoman over every woman, so that rather than allowing women to be who they are, it has created this vision of who they need to be. This is not real freedom, it is merely a submission to a self-created icon – an exercise more in tribal religion that in socio-political ideology. Didion writes: "All one's actual apprehension of what it is like to be a woman, the irreconcilable difference of it – that sense of living one's deepest life underwater, that dark involvement with blood and birth and death – could now be declared invalid, unnecessary, one never felt it at all."(italics in the original). Or elsewhere: "The astral discontent with actual lives, actual men, the denial of the real generative possibilities of adult sexual life, somehow touches beyond words….These are converts who want not a revolution but 'romance', who believe not in the oppression of women but in their own chances for a new life exactly in the mold of their old life. In certain ways they tell us sadder things about what the culture has done to them than the theorists ever did, and they also tell us, I suspect, that the movement is no longer a cause but a symptom." Simply brilliant.
There are many other interesting essays here, and the writing is uniformly excellent, so that reading The White Album is a real treat. Don't miss it.
To close, one last quote that I can't resist posting:
"I could not visit my mother-in-law without averting my eyes from a framed verse, a "house blessing' which hung in a hallway of her house in West Hartford, Connecticut.
God bless the corners of this house,
And be the lintel blest –
And bless the hearth and bless the board
And bless each place of rest –
And bless the crystal windowpane that lets the starlight in
And bless each door that opens wide, to stranger as to kin.
This verse had on me the effect of a physical chill, so insistently did it seem the kind of 'ironic' detail the reporters would seize upon, the morning the bodies were found."
 In the interest of fending off indignant comments, let me say that I freely admit to not being an expert on the movement, and am aware that there are many ways in which Didion's critique is invalid in the 21st century. I still think, however, that for all the wealth of opportunities the movement has created for women, it has not managed to rid itself (and them) of the pressure to utilise and conform to these opportunities. The need to match up to some predefined ideal hasn't gone away; it's just that that image has changed and become harder to live up to.


- An adaptable system
- A removable housing
- Two elements to connect
- A smart rain gauge
- Place the funnel
- Position the detector
- Adjust the system according to the nature of the soil
- Plant the probe
Whatever the size of the garden, automatic watering is a definite plus. Finished the chores, place to the comfort...
An adaptable system
Automatic watering can bring the right amount of water to your plants, at the right time, even in your absence.
A humidity sensor and a rain gauge, two accessories to connect directly to the programmer, can further improve watering. The first is planted in the ground and cuts the watering when the ground is already too wet.
If you live in an area where, at certain times of the year, morning dew is enough to water your plants, this device is ideal.
The rain gauge will pause the watering.
Both devices save water and avoid drowning your plantations.
A removable housing
On some programmers (as here on this model), the front panel is detachable, which simplifies the settings.
Two elements to connect
At the rear of the irrigation programmer is a connection terminal for connecting the rain gauge or humidity sensor.
A smart rain gauge
The rain gauge consists of a cell in which a light beam circulates. In the presence of moisture, the beam is deflected, watering is suspended.
Place the funnel
Position the funnel that collects the raindrops if you want watering to be cut at the slightest rainfall. Remove it to water more abundantly.
Position the detector
Place the detector in a location where it will not be protected from rain but where it will not receive water from watering.
Adjust the system according to the nature of the soil
Adjust the sensitivity according to the nature of the soil: high sensitivity (sandy soils), low (flowering lands), medium (non-forest soils, clay humus).
Plant the probe
Turn the soil over 12 cm to loosen the soil and remove the stones. Dip the end of the probe into the water and then plant it to its base.


text:
Keanu Charles Reeves (September 2, 1964) is a Canadian actor. Reeves, who was raised in Toronto but was born in Beirut, began his acting career in theatre and television before making his big-screen debut in Youngblood (1986).
Since then, he has repeated his role in Bill & Ted's Excellent Adventure and its sequels. In 1991, he received critical acclaim for his performance as a hoodlum in the indie movie My Own Private Idaho, and he went on to star in the action films Point Break and Speed two years later (1994).
In The Devil's Advocate (1997), following a string of box office disappointments, Reeves's performance was positively appreciated. In 1999, he became even more famous for his role as Neo in the science fiction series The Matrix. As John Constantine, he starred in Constantine (2005).
He has also appeared in the romance drama The Lake House (2006), the science-fiction thriller The Day the Earth Stood Still (2008), and the criminal thriller Street Kings (2009). (2008). Reeves made a return in 2014 as the eponymous assassin in the John Wick film series, following several years of critically and commercially underwhelming performances.
In addition to starring in the film Man of Tai Chi, Reeves has also directed it (2013). He was a member of the rock band Dogstar and has also dabbled in writing and charity.
Keanu On September 2, 1964, in Beirut, Lebanon, Charles Reeves was born. He was born to a mother from Essex in the United Kingdom and a father from Oahu in the United States. While working as a costume designer in Beirut, Reeves' mother met his father.
It wasn't until he was a teenager that the family settled down in Toronto and became Canadian citizens after his father left the family when he was just three years old.
Reeves' schooling was hampered by his dyslexia. A number of high schools kicked him out. However, as a goalkeeper for his high school hockey team, he made up for his poor academic performance. For the first time in his life, Reeves decided to give up hockey and education in order to pursue a career in acting.
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Reeves got his start in the entertainment industry doing bit parts on Canadian television, in commercials, and in a variety of stage performances before making his debut in the feature film Youngblood in the year 1986.
After that, he was offered other roles as a co-star, one of which being a more significant part in the film River's Edge, which received positive reviews. Bill and Ted's Excellent Adventure was a surprising triumph at the movie office in 1989, grossing more than $40 million in the United States alone.
This success was Reeves' first taste of commercial success. After that, Reeves went on to feature in a plethora of films, both big and independent, some of which include the drama “Point Break” and the independent “cult smash” “My Own Private Idaho.”
Reeves was catapulted to A-list prominence as a result of the popularity of the action film Speed, which was released in 1994. The film was nominated for two Academy Awards and grossed a combined total of $350.5 million domestically and internationally.
Further success came to Reeves as a result of his leading role in The Matrix franchise, which consists of three science fiction action pictures with a combined gross income of $1.6 billion dollars. His performance as Neo was lauded and commended, and audiences went wild for the flicks thanks to its star.
In the 2014 action thriller “John Wick,” in which Reeves assumed the role of a retired assassin, he was cast in the lead role of the film's titular character. Both the critical reception and financial success of the films were quite positive. He went on to play the same character in two more films after the first one.
More than $500 million has been made at the box office as a direct result of his John Wick series. After taking into account the effects of inflation, Keanu's films have together earned more than $4.6 billion at theatres throughout the world as of the time of this writing.
The first-ever Keanu Reeves film festival in Europe was held in Glasgow, Scotland in 2019, and it was called “KeanuCon.” The event took place on the 27th and 28th of April, and during the course of those two days, nine of his movies were shown.
Keanu Reeves's Net Worth
Keanu Reeves is a well-known and very successful actor, singer, and producer in addition to being a generous philanthropist. His net worth is estimated at $380 million.
Over the course of his career, he has been in scores of films, some of which have been commercially successful to the tune of billions of dollars. Perhaps his most notable contributions are those he made to the Matrix and John Wick film franchises.
Keanu Reeves Earnings from The Matrix
Will Smith declined the opportunity to play Neo in The Matrix, which led to Keanu Reeves being cast in the role instead. His starting salary was going to be ten million dollars. He also was given a chunk of the backend which, thanks to the movie's surprise success, resulted in total earnings of $35 million for the first movie.
Taking into account the effects of inflation, that is equivalent to almost $53 million. Warner Bros. Pictures made the announcement that they will be producing a fourth Matrix film in August of 2019.
Keanu Reeves' backend bonuses would eventually make him the highest-paid actor in the history of the film industry for a single franchise if you factor in DVD sales (The Matrix was one of the best-selling DVDs of all time), as well as the enormous box office success of the second and third movies in the series.
Keanu received an additional minimum of 120 million dollars in bonuses from the production of the second and third films. As of the time this article was written, his total revenues from The Matrix, including all sources, have surpassed $200 million.
On December 24, 1999, Jennifer Syme, Reeves' longtime girlfriend, gave birth to their stillborn daughter, Ava Archer Syme-Reeves. Their sadness harmed their relationship, and they broke up a few Syme died in a vehicle accident in April 2001. Reeves wanted to cope with the situation before starting filming The Matrix sequels back-to-back. Dat China Chow in 2008. He's never married and very private about his personal life.
Reeves is not religious but is spiritual and is interested in Buddhism.
Two stalkers visited Keanu's Hollywood Hills house in 2014. On September 12, he awakened to a stalker in his library waiting to see him. In the end, he phoned the cops. Another stalker came three days later through an open fence left by Reeves' cleaners. The stalker undressed, showered and then swam naked in his pool. When Reeves wasn't home, the cleaning team called the cops, who arrested the stalker.
Frequently Asked Questions
Who Was Keanu Reeves's Wife?
An American actress, personal assistant, and record business executive, Jennifer Maria Syme (December 7, 1972 — April 2, 2001) was known for her work in the entertainment industry.
What Does Keanu Reeves Do With His Money?
He has a lengthy history of giving money to crew workers who work on his films. On top of all the money that Reeves has donated to cancer research and his personal foundation, Reeves has gone out of his way to guarantee that crew workers on his films are well taken care of.

domain: entertainment


TE-Z8 is a simple OBD controller, designed to work in tandem with Tegas Engineering gas controllers . By connecting to the diagnostic bus, it requests data about engine parameters and converts into an analogue output signal. TE-Z8 output is connected to the gas controller lambda wire. Lambda is not connected.
Provided two types of data:
1) Composite signal output of the short and long trim (petrol engine)
2) Calculated value output of the load on the engine (diesel engine).
Type of data selection is done by setting jumpers inside the housing.
KWP2000 slow, KWP2000 fast, ISO 9141 (K-Line)


On The Quarter Deck
Scottish Country Dance InstructionON THE QUARTER DECK (R8x32) 2C (4C set) Iain Boyd Harbour City
1- 8 1s set and cross over RH, set and ¾ turn RH
9-16 1s dance RH across with 2M and LH across with 2L
17-24 1L followed by 1M dance down for 2 and 1M followed by1L dance up for 2, both cast on Ladies' side and 1M crosses to 2nd place own side
25-32 2s+1s R&L
(MINICRIB. Dance crib compiled by Charles Upton, Deeside Caledonian Society, and his successors)
Keith Rose's Crib Diagrams
Dance Instruction VideosOn The Quarter Deck - Scottish Country Dancing Instruction Video
Dance InformationA deck is a permanent covering over a compartment or a hull of a ship.
The Quarterdeck can mean either the part of the upper deck behind (abaft) the mainmast, including the poop deck when there is one, usually reserved for ship's officers, guests, and passengers. Also it can mean the area to which a gangway for officers and diplomatic guests to board the vessel leads. Also any entry point for personnel.
Illustration Of The Quarter Deck Of An 18th Century Frigate
Dance information licensed under this Creative Commons Licence 3.0.
Text from this original Quarter-deck article on Wikipedia.
Image copyright (cropped) Illustrator unknown [Public domain], via Wikimedia Commons.
Back to the top of this Scottish Country Dancing Instructions 'On The Quarter Deck' page


Memory Dual Channel not detected - Printable Version
+- HWiNFO/32/64 Forum (http://www.hwinfo.com/forum)
+-- Forum: Bug Reports (/Forum-Bug-Reports)
+--- Forum: HWiNFO32 & HWiNFO64 Bug Reports (/Forum-HWiNFO32-HWiNFO64-Bug-Reports)
+--- Thread: Memory Dual Channel not detected (/Thread-Memory-Dual-Channel-not-detected)
Memory Dual Channel not detected - city_zen - 03-11-2010 08:22 PM
HWiNFO32 is my favorite hardware inventory program. Thanks for developing it.
There is just one bug that seems to keep showing up and it's the fact that my system's memory keeps getting detected as running in Single Channel, while CPU-Z correctly detects as running in Dual Channel.
I'm attaching a screenshot.
Let me know if you need more info.
Re: Memory Dual Channel not detected - Martin - 03-11-2010 08:49 PM
thanks for your report.
Yes, I need more info - please attach a Report + Debug File.
See http://www.hwinfo.com/forum/Thread-IMPORTANT-Read-this-before-submitting-a-report for instructions how to create a Debug File.
Re: Memory Dual Channel not detected - VictorVG - 03-11-2010 09:40 PM
That is chip set and hardware mode using Your motherboard? Some combination CPU+Motherboard+Memory chip can't stable work on dual channel mode, also all DRAM/SRAM memory chip use multi matrix architecture for minimize hardware delay timings and CPY-Z can indicate as dual channel simple memory chip bus. Also on Your report I can't read the CPU type - as samples, if Your use AMD Socket-754 CPU, then memory controller on this CPU work's only on single channel mode. On Your report I can't read this technical data. Please check for control at memory controller real use Dual Channel mode the over hardware information tools, example Lavalys Everest (AIDA32) Ultimate Edition. If use this tool on trial mode don't create report, but screen information Your can use for control.
Also Your can use any my bug reports as examples - viewtopic.php?f=5&t=13
Best regards, Victor
Re: Memory Dual Channel not detected - city_zen - 03-12-2010 01:22 AM
OK, I'm attaching a rar file which contains a report and a debug file.
Let me know if you need more info or if I can help you debug it in any other way.
Re: Memory Dual Channel not detected - city_zen - 03-12-2010 01:31 AM
VictorVG Wrote:city_zenMy system's specs:
- CPU: Intel Core 2 Duo E6550 2.33 GHz
- Memory: 2 x 1 GB DDR2 PC2-6400 (400 MHz)
- Motherboard/Chipset: MSI MS-7345 / Intel P35 + ICH9R
Both the CPU and the memory are overclocked.
I'm fairly sure I've seen the legend "Dual Channel" on the screen during the computer's boot process, that's why I think CPU-Z is giving the right info and HWiNFO32 is not. Plus, my two memory sticks are identical and installed in alternated slots on the motherboard (as they are supposed to go in order to work in dual channel). We'll see.
Re: Memory Dual Channel not detected - Martin - 03-12-2010 08:03 AM
Thanks for the additional data. Now I can see that this is a bug in HWiNFO32.
Please try this build: http://www.hwinfo.sk/beta/hw32_343_636.zip and let me know if it's fixed now.
Re: Memory Dual Channel not detected - city_zen - 03-12-2010 06:25 PM
Martin Wrote:Thanks for the additional data. Now I can see that this is a bug in HWiNFO32.
HWiNFO32 now detects my memory as running in Dual-Channel.
Thanks a lot for fixing it.
Re: Memory Dual Channel not detected - Martin - 03-12-2010 07:43 PM
You're welcome. Thanks for the feedback.
city_zen Wrote:It's fixed!!!
Re: Memory Dual Channel not detected - VictorVG - 03-15-2010 01:30 AM
city_zen Wrote:My system's specs:Your system is far beyond the stability of the of logic circuits, and you have caused a significant decrease in reliability due to the sharp, in the tens of thousands of aging accelerate aging of the semiconductor. Nevertheless, this is your personal right - the system fails after six months or a year - to buy a new systems and pay for data recovery from damaged disks - the bus frequency has increased over the admissible values by 100% leads to failure of devices. It's me you like IT system engineer with practical experience of over 30 years and I can guarantee it. You can take a risk, but the report says that the system works in an invalid mode. Do you think we, as developers of equipment for nothing lay in its provision for operating frequencies and waveform signals? Scatter parameters even with chips per wafer is in the range of 15% - 20%, and from different parties and may reach 30% - 40%.
Re: Memory Dual Channel not detected - NeoCat - 04-11-2010 09:46 PM
bug still active in latest version of hwinfo32 (3.45-674)
cpu: core 2 duo e6320
m/b: biostar tforce p965
ram: 2x1gb kit ocz xtc soe pc2-6400
Re: Memory Dual Channel not detected - Martin - 04-11-2010 10:00 PM
Thanks for the report. The previous fix was for other chipsets, now I extend it to P96x.
Please try this build: http://www.hwinfo.sk/beta/hw32_345_682.zip
and give feedback.
NeoCat Wrote:bug still active in latest version of hwinfo32 (3.45-674)
Re: Memory Dual Channel not detected - NeoCat - 04-12-2010 10:45 AM
Martin Wrote:Please try this build: http://www.hwinfo.sk/beta/hw32_345_682.zipThanks, now displays correctly


text:
Brie ♥ Apricot
À la Monsieur
Soft Thinbread Roll with Pickled Carrots, Fresh Sprouts and Hummus
Thaw & Serve. Our bread thaws in just a few minutes. A good way to reduce waste!
Soft Thinbread Roll with Grilled Chicken, Wheatberries and Pea Spread
Soft Thinbread Roll with Smoked Whitefish, Potatoes and Browned Butter
Soft Thinbread with Beans, Potatoes and Nuts
Lunch box tray – Smoked ham, cheese, honey and cornichons
Lunch box tray – Chorizo, rucola and corn
Snacking tray – Smoked salmon
Did you see our concept Mini Rolls? Small, delicious bites with Thin Bread.
Snacking tray – Avocado, tofu and dill
Sharing tray – Grilled bell pepper, turkey and barbeque sauce
Sharing tray – Smoked salmon
Wrap with spicy sausage, coleslaw, courgette and pickled red cabbage
Caesar salad with prawns and hot-smoked salmon in a wrap
Malmö + Meze
Check out our concept Wrapini. A small Thin Bread Roll to stay or go.
Snacking tray – Hummus, feta cheese and pomegranate
Sharing tray – Kale, ginger and wasabi
Lunch box tray – Salmon, nori, avocado and sriracha mayonnaise
Vegan Colour Bliss
Eggstra Good Morning


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Going Down Home With Daddy
Author: Kelly Starling Lyons
Illustrator: Daniel Minters
Ages: 4 to 8 years old, Grades PK to 3
When we go down home with Daddy, everything we see holds a piece of him and us. We head up the highway thinking about family and dreaming about next year.
–Kelly Starling Lyons
I’ve never experienced a family reunion. My family is scattered to the wind. When my parents emigrated from Cuba, years before Fidel Castro came to power, they left behind the bulk of our family; and, like seeds from a dandelion flower, floated gently until they settled in the United States. I have a large family. I just don’t know most of them. And, I never see them.
Books like Lyons’ Going Down Home With Daddy, are a mystery to me. A dad piles his family into a car and they drive down home to see great-grandma Granny and share in an amazing anniversary celebration where everybody shares something personal. Sounds amazing. And it is!
Lyons has given me the opportunity to attend a family reunion, like one I’ve never experienced. Her characters come alive on the page, even in a scant picture book setting. We are visiting Daddy’s family, at the family home where the land stretches as far as the eye can see. With nothing more than a couple of sentences each, sometimes a single sentence, Lyons has the ability to define a character so clearly in your mind, that you can hear him as you read.
‘Got a head just like your daddy,’ Uncle Jay teases me.
Daddy’s eyes twinkle. ‘Now I know you’re not talking about heads.’
‘Can’t take them anywhere.’ Grandma Loretta says laughing.Going Down Home With Daddy
Lyons’ ear for dialogue is exceptional. Her dialogue rings true to life and immerses you in the family gathering–from the gentle ribbing between cousins to the laughing and giggling that is easily present at a family reunion. There is, a poetic nature to her prose, that shimmers in the details she provides. Take for instance:
‘Cotton for the quilts Granny made to keep her children warm,’ I say holding a white cloud in my fingers.
‘A pecan for the trees Pa planted and all of the kids love to climb.’
I pinch dirt and let it rain to the ground. ‘And earth for land that’s ours as far as we can see.’Going Down Home With Daddy
Minter’s illustrations are breathtaking and resemble a cross between vibrant watercolors and hard line paintings. They are masterful, employing a bright yellow and mellow blue palette reminiscent of southern jazz. It is no wonder that Minter received a Caldecott Honor for his illustrations of this book. The devil is in the details and Minter spares none, brandishing interesting patterns where you wouldn’t expect them and layering layer upon layer of soft tones to enhance gorgeous backgrounds.
This is a unique book. You get a peek into the lives of a multi-generational family and experience the bond between father and son. Gorgeous, poetic prose and beautiful illustrations. What more could you possibly want?
More books for this Age Group can be found here.
Please, leave comments! I love a HEALTHY exchange of ideas. After all, critical thinking is essential to life.
My thanks to Peachtree Publishing for providing a complimentary copy of this book. The views expressed herein are my own.
*As an Amazon Associate I earn from qualifying purchases.


Iranian foreign ministry spokesman has hailed the results of Austria's parliamentary elections and the victory of People's Party led by incumbent Foreign Minister Sebastian Kurz.
Iran's foreign ministry spokesman says a delegation of Canadian foreign ministry officials has recently visited Tehran and held positive talks with Iranian officials.
In a Monday statement, Foreign Ministry Spokesman Bahram Qassemi said Tehran welcomes the results of Kyrgyzstan's presidential votes, and is prepared to further promote...
Uzbekistan's Foreign Minister Abdulaziz Kamilov will arrive in Tehran later on Monday to hold talks with high-raking Iranian officials, Iran’s Foreign Ministry Spokesman Bahram Qassemi announced.
A senior advisor to Iran’s president says Trump’s comments will only increase the Iranian people’s support for the Islamic Revolution Guards Corps (IRGC).
Iranian parliament speaker has once again slammed US president’s anti-Iran rhetoric, saying Donald Trump lacks a systematic view of the global peace and stability.
Iranian media have continued to show strong reactions to Donald Trump’s comments against Iran and the Iran nuclear deal.
Iran has denied reports that the country has closed its land border with the semi-autonomous Iraqi Kurdistan Region in response to last month’s independence referendum in the region.
Iran’s Parliament Speaker Ali Larijani says US President Donald Trump’s recent anti-Iran speech showed strong traces of lobbying by the Israeli regime and Saudi Arabia.
British Foreign Secretary Boris Johnson and Iran's Foreign Minister Mohammad Javad Zarif have held phone talks on the nuclear deal between Tehran and world powers, also known as JCPOA.


text:
Judged by a panel of industry experts, the annual European Hotel Design Awards celebrates newly opened European hotels that showcase exceptional construction methods and interiors. After visiting each shortlisted property, the panel meet to debate their merits and select their favourites. From the best renovation to the best spa, here are the winners of the 2015 European Hotel Design Awards
The Beaumont Hotel
Headquartered in London’s swanky Mayfair district, Beaumont Hotel is the first foray into luxury lodging by restaurateurs Chris Corbin and Jeremy King. While the Art Deco interior echoes the building’s history, the judges particularly cited the hotel’s boldly modern, three-story Cubist-style suite as a “genius” touch.
Mandarin Oriental Bodrum
Set amid 150 hilltop acres overlooking the gleaming Mediterranean, the Mandarin Oriental Bodrum is the hotel group’s first property in Turkey. The rooms and public spaces were designed by Antonio Citterio to blur the boundaries between inside and out, especially in the suites and villas, which have their own private plunge pools and terraces.
The Peninsula Paris
The Peninsula Paris poured 580 million dollars into the four-year restoration of the 1908 Belle Époque building where George Gershwin composed “An American in Paris” and the Paris Peace Accords were signed. Judges praised the meticulous and imaginative update that resulted in 200 luxurious rooms, as well as the lower-level spa and indoor pool, and private rooftop terraces.
Les Bains‘ storied past began in 1885, when it opened as a Parisian bathhouse. By the 1970s it was a star-studded nightclub-cum-pool-party—envisioned by the then-unknown Philippe Starck—before closing in 2010. A four-year renovation downsized the bar in favor of adding 39 rooms, designed by Tristan Auer, that pay homage to those glamorous days and feature red-velvet sofas like the one Andy Warhol had in his studio. Not only did Les Bains earn praises from the judges for its “lived-in local narrative,” but it also captured the Hotel Design of the Year award for being “simply breathtaking from every angle.”
INK Hotel Amsterdam
Clues to its past are nearly everywhere you look at INK Hotel Amsterdam. This boutique property was once the headquarters of Dutch newspaper De Tijd, and the contest’s judges were impressed by the way architects at Concrete, a local firm, had “created a lively and welcoming lobby clearly informed by the building’s historic past use.” Oversize typeset letters decorate the walls of the lobby, while the library lounge is covered with newspaper-plate “negatives”—a nostalgic throwback to pre-digital days.
Denis Montel of RDAI, responsible for designing Hermès boutiques around the globe, was commissioned to bring his vision to La Salle-à-Manger, the restaurant at Paris’s Les Bains hotel. Judges declared it “a memorable, sexy, and enchanting restaurant.” The dramatic ceiling, sculpted in undulating domes of dark red-lacquer that seem to melt down the walls and supporting columns, is softened by a fabric-covered wall of gradient tones. Judges also gave a nod to Le Reservoir, the private dining room that was once a 50-foot-tall water tank.
Mandarin Oriental Bodrum – Spa
The expansive Spa at Mandarin Oriental Bodrum expands upon the hotel’s guiding philosophy that, in a setting such as this, design shouldn’t detract from nature. The judges took note of how perfectly the Turkish retreat integrated indoor and outdoor environments, including private cabanas with sprawling terraces, an 85-foot indoor pool as well as several outdoor vitality pools, and open-air lounging areas.
Le Bar at Hôtel Plaza Athénée
Architecture firm Jouin Manku was aiming for something quite different than the traditional hotel bar rife with antiques and crystal when designing Le Bar at Hôtel Plaza Athénée, and the judges commended him for it. In the early evening—underneath a cloud of sapphire fabric (more than 500 yards)—a faint blue light suffuses the space, suggesting a restless, stormy sky. The bar itself is a sweeping arc of clear resin mixed with streaks of white paint, giving it the appearance of clouds. But come at 11 p.m., when the room is bathed in red light as a DJ spins house music and Paris’s well-heeled club crowd takes over.
Garden House Suite at Rosewood London
The rooftop Garden House Suite at Rosewood London owes its name to the terrace accessed through the den, providing an oasis of greenery as well as picturesque views over the courtyard below and the city’s rooftops beyond. Inside, the suite invites comparisons to a fine manor house, with its living room, library, den, Italian-marble bathroom, powder room, and private service entrance. Judges called it an urban sanctuary that “takes the trophy for its luxurious residential feeling.”


text:
Ariton opens 2020 season at home against Dale County
At long last the 2020 high school football season will get underway for the Ariton Purple Cats on Friday night when they host Dale County at Robert F. Zumstien Stadium.
For the third consecutive season the Purple Cats will open the season against the Dale County Warriors.
“This game is going to help us win or lose,” head coach Steven Kilcrease said. “Playing that type team, it’s going to be a physical game. I think it’s going to be a challenge for us, but I like coming out of the gates playing good teams.”
The Purple Cats have fallen to the Warriors in each of the last two season openers, but ultimately Kilcrease believes those games have helped springboard his team to future success.
“It has made us better each year,” Kilcrease said. “Last year was a close game, but they put it to us late in the game. I think it’s a challenge, but I think it prepares us for region play.”
Robert F. Zumstein Stadium will have limited capacity beginning on Friday night. The number of tickets that will be sold will be 750 but that will not include reserved seats or seats reserved for the band. Players on both teams as well as cheerleaders and band members will each be given 2 tickets. Anything left over after that will be general admission. Gates will open at 5:30 p.m.
“If it weren’t for the restrictions this place would have people falling over the gates to get in,” Kilcrease said. “Everybody is ready to get out and do something. We are having to cut back on the number of tickets we can sell but that’s where we are right now. The community is excited.”
The Purple Cats, like many other schools, have had limited amount of time to get ready for the opening. Ariton has spent very little time doing much tackling this summer. Because of that there will be some question marks when the game kicks off on Friday night.
“I don’t know a coach that likes the first game,” Kilcrease said. “There is always an element of surprise. You never know what you’re going to see. It’s exciting, but at the same time, do you have everything covered?”
Due to the threat of COVID-19, there will be an added challenge this year.
“There are a lot of things as a head coach that you worry about,” Kilcrease said. “We are just ready for a game, we are ready to play. We try to not have to worry about all that. We want to worry only about football.”
Kickoff is set for 7 p.m.