HIV-1 establishes latency primarily by infecting activated CD4+ T cells that later return to quiescence as memory cells. Latency allows HIV-1 to evade immune responses and to persist during antiretroviral therapy, which represents an important problem in clinical practice. The lack of a valid cellular model to study HIV-1 latency has hindered advances in the understanding of its biology. In this study, we attempted to model HIV-1 latency using human primary CD4+ T cells infected in vitro with HIV-1 after activation with Ag-loaded dendritic cells and then brought back to quiescence through a resting phase in the presence of IL-7. During the resting phase, expression of cellular activation markers disappeared and cell proliferation and viral replication ceased, but resumed following restimulation of rested cells with Ag or mAbs directed to CD3/CD28. In addition, higher cell death rates were observed in HIV-1-infected than uninfected cultures during secondary but not primary stimulation. Thus, this system may allow us to study the biology of HIV-1 latency, as well as the mechanisms of CD4+ T cell death following HIV-1 reactivation.
Although highly active antiretroviral therapy (HAART)2 suppresses viral replication to undetectable levels, HIV-1 continuously replicates throughout the course of the infection (1). However, HIV-1 also establishes a status of nonproductive infection known as viral latency. Harper et al. (2) reported that cells harboring the provirus in clinical samples were found at frequency considerably higher than cells expressing viral RNA, thus providing the first indirect evidence for the existence of latently infected cells in HIV-1 patients. Formal demonstration came from Siliciano and colleagues (3), who detected integrated, latent HIV-1 genome in resting peripheral blood CD4+ T cells of HIV-1 patients. Latently infected cells persist in HAART-treated patients with undetectable viremia, and HIV-1 can be rescued in vitro from the cells of these patients (4, 5, 6). The rate of decay for the latently infected pool has been calculated at 44 mo, sufficiently slow to guarantee lifelong persistence and to preclude HIV-1 eradication with HAART (7). Moreover, as a dormant provirus, HIV-1 is invisible to cellular and humoral immune responses and resistant to drugs interfering with its life cycle (8).
HIV-1 establishes latency by exploiting the cellular events of CD4+ T cell blast transformation, clonal expansion, and clonal contraction that lead to the formation of immunological memory (for a comprehensive review, see Refs. 9 and 10). During the contraction phase at the end of the immune response, the vast majority of activated CD4+ T cells die in response to intrinsic mechanisms triggered by Ag withdrawal and consequent decline of growth factors and Bcl-2, as well as extrinsic mechanisms involving Fas (CD95)-Fas ligand (CD178) interaction and caspase 3 activation (11). Production of IL-7 during the contraction phase promotes the establishment and maintenance of immunological memory through the up-regulation of Bcl-2 expression, which counteracts the intrinsic pathway of activated CD4+ T cell apoptosis (10, 12, 13). HIV-1 typically infects cells during blast transformation and clonal expansion (14, 15). Although the majority of HIV-1-infected lymphoblast CD4 cells die as a consequence of apoptosis or cytopathic effects of HIV-1, some infected cells survive clonal contraction and return to the resting state as memory cells bearing an integrated HIV-1 genome. In their resting state, HIV-1-infected cells do not actively replicate the virus, which remains dormant for extended periods of time (16, 17). Viral production resumes following Ag-driven or homeostatic proliferation and cytokine stimulation.
Currently, the study of HIV-1 latency relies primarily on clinical samples and chronically infected cell lines, which present severe limitations such as the very low frequency of latently infected cells in the former and the lack of physiological relevance of the latter (for a review see Ref. 8). In addition, systems based on the use of clinical samples allow us to study HIV-1 reactivation from latency, but not how latency is established. In this study, we provide proof of concept that resting nonproductively infected CD4+ T cells carrying a replication-competent HIV-1 genome can be generated in vitro after Ag-driven proliferation. This system may help shed light into the mechanisms of HIV-1 latency and lead to identify new therapeutic strategies.
Peripheral blood was obtained from HIV-1-negative volunteers after approval by the Institutional Review Board of the University of Maryland, Baltimore, and with signed informed consent forms. PBMC were isolated by Ficoll-Paque (GE Healthcare) centrifugation and washed three times in PBS (Invitrogen).
Monocytes were enriched by negative selection from freshly isolated PBMC using a Monocyte Isolation Kit (Miltenyi Biotec). Enriched monocytes were suspended in RPMI 1640 (Invitrogen) and subjected to a second round of enrichment by adhesion on plastic at 37°C. After 2 h, cultures were washed to remove nonadherent cells. For the generation of immature monocyte-derived dendritic cells (MDDC), adherent cells were cultured for 5 days in MDDC medium consisting of RPMI 1640 supplemented with 1% nonessential amino acids, 1% sodium pyruvate, 50 μM 2-ME, 50 μg/ml gentamicin (all from Invitrogen), 10% human serum AB (Gemini Bioproducts), 50 ng/ml GM-CSF and 50 ng/ml IL-4 (both from R&D Systems), with a 50% medium change at day 3. Monocytes and MDDC were analyzed by flow cytometry (FCM) using anti-CD14, CD86, and HLA-DR Abs (see below).
Naive CD4+ T cells were enriched by negative selection from freshly isolated PBMC using a naive CD4+ T cell isolation kit (Miltenyi Biotec). Enriched cells were mixed with MDDC at a 10:1 ratio and cultured in MDDC medium containing 250 ng/ml staphylococcal enterotoxin B (SEB; Sigma-Aldrich) and 25 U/ml IL-2 (Roche Biochemicals). After 4 days, activated cells were harvested, washed, and suspended in RPMI 1640 and then infected with 2 × 103 tissue culture infective dose 50 per 106 cells of gradient-purified HIV-1IIIB (Applied Biosystems) for 2 h at 37°C. Finally, the cells were washed three times to remove cell-free virus, plated at 0.5 × 106 cells/ml in fresh MDDC medium containing 25 U/ml IL-2, and cultured for 10–15 days. Every 2–3 days, fresh MDDC medium plus IL-2 was added to bring the cell concentration back to 0.5 × 106 cells/ml. At each step, cells were analyzed by FCM with anti-CD3, CD4, CD25, CD45R0, CD45RA, CD62L, CD69, HLA-DR, and Ki-67 Abs. In addition, diploidy was analyzed by intracellular staining with 7-aminoactinomycin D (7AAD; BD Biosciences).
Memory CD4+ T cells were enriched by negative selection from 10- to 15-day cultures of HIV-1-infected and uninfected CD4+ T lymphoblasts using a memory CD4+ T cell isolation kit (Miltenyi Biotec). Highly enriched cells were then cultured in RPMI 1640 supplemented with 10% human serum AB and 1 ng/ml IL-7 (R&D Systems) for up to 30 days. For reactivation of HIV-1 replication, rested cells were stimulated with SEB-loaded dendritic cells as described above. Alternatively, rested cells were plated in tissue culture wells coated overnight with 1 μg/ml anti-CD3 (OKT3) and CD28 (CD28.2; Beckman Coulter) in PBS at 4°C and cultured in the presence of 25 U/ml IL-2. Where indicated, cells were cultured in the presence of 10 μg/ml cycloheximide (CHX; Sigma-Aldrich) or 1 μM azidothymidine (AZT; Sigma-Aldrich).
For surface staining, 2.5 × 105 cells were washed with 1 ml of 2% human serum AB in PBS and suspended in 200 μl of the same buffer containing 2–5 μl of the appropriate CD-specific or isotype control Ab. After 30 min on ice in the dark, cells were washed twice with the same buffer and fixed in 1% paraformaldehyde (Sigma-Aldrich) in PBS. For intracellular detection of HIV-1, we used the anti-p24gag KC57 Ab (Beckman Coulter) according to the instructions of the manufacturer. For intracellular detection of Ki-67 and determination of diploidy, cells were fixed and permeabilized with a Cytofix/Cytoperm Kit from BD Biosciences, then stained with the appropriate Ab or with 7AAD. All Abs were from BD Biosciences, except for the anti-p24gag KC57 Ab, which was obtained from Beckman Coulter. Stained cells were acquired with a FACSCalibur flow cytometer using CellQuest software (both from BD Biosciences). FCM data were analyzed with FlowJo (Tree Star).
For extracellular HIV-1 p24gag Ag ELISA, aliquots of the culture supernatant were collected, rendered 0.5% with Triton X-100 and 0.002% with sodium azide, and stored at −20°C until analysis. For intracellular ELISA, 105 cells were harvested, washed with PBS, pelleted, and lysed in 0.5% Triton X-100/0.002% sodium azide in PBS and stored at −20°C until analysis. Samples were then analyzed using an ELISA kit from NEN Life Sciences (PerkinElmer). The lower limit of detection of this kit is 6.25 pg/ml. All ELISA were performed by the μQuant Facility of the Institute of Human Virology.
The model of HIV-1 latency we have developed recapitulates in vitro the events of blast transformation, clonal expansion, and clonal contraction leading to the generation of resting, memory cells. It consists of four steps (Fig. 1⇓A): 1) generation of dendritic cells from primary monocytes (MDDC), 2) activation of naive CD4+ T cells with MDDC and Ag, 3) HIV-1 infection and expansion of activated CD4+ T cells, and 4) generation of resting, nonproductively infected cells. The activation and expansion of naive CD4+ T cells with MDDC is a modification of a published method (18). A fifth step involves the reactivation (secondary stimulation) of resting, nonproductively infected cells with either MDDC plus SEB or immobilized anti-CD3/CD28 Abs leading to rescue of latent provirus. For consistency, all results shown below were obtained with cells from the same individual donor in the course of the same experiment (unless otherwise indicated) and are representative of results obtained with more than five donors.
Enrichment of naive CD4+ T cells and activation with dendritic cells. A, Experimental procedure for the generation of resting nonproductively, infected CD4+ T cells. B, FCM analysis of naive CD4+ T cells enriched by negative selection from PBMC. C, FCM analysis of CD4+ T cells after activation with MDDC. All plots (except for forward light scatter height (FSC-H) vs side light scatter height (SSC-H)) are gated in the live cell population as determined by FSC and SSC profiles and exclusion of 7AAD. The days of analysis reported in both panels match the time scale reported in A. Results are representative of more than 20 different donors.
First, we isolated monocytes by negative selection from fresh PBMC of HIV-1-negative donors. These cells expressed high CD14 and low CD86 (data not shown), indicating strong Ag capture function, but poor Ag presentation and T cell costimulation function. Following a further enrichment step by adherence to eliminate contaminating lymphocytes, we induced differentiation of adherent monocytes into dendritic cells (MDDC) with IL-4 and GM-CSF. Immature MDDC displayed increased size and granularity, low CD14 and high CD86 expression (data not shown), and thus possessed strong Ag presentation and T cell costimulation properties.
Next, we enriched naive CD4+ T cells by negative selection from fresh PBMC of the same donor, yielding naive cells at a purity consistently ≥95% (Fig. 1⇑B). As expected, these cells did not express activation markers and displayed a diploid genomic DNA content, indicating absence of ongoing DNA synthesis (Fig. 1⇑B). All preparations of naive CD4+ T cells contained a small population of CD25+ cells (median frequency, 4%; minimal, 1%; maximum, 10%; n = 13). These cells expressed lower CD127 levels compared with the main CD25− population and were dimly positive for the transcription factor FoxP3 (data not shown). Based on these features, we concluded that the population of CD25+ cells contained CD4+CD45RA+CD127lowFoxP3low natural naive regulatory cells described previously (19, 20). Naive cells were activated by coculture with autologous MDDC and Ag. To achieve T cell responses of sufficient magnitude to sustain measurable levels of HIV-1 replication, we used the superantigen SEB instead of nominal protein Ags. Fig. 1⇑C demonstrates that by day 4 after stimulation (that is day 9 of the entire procedure according to the time scale shown in Fig. 1⇑A), a high percentage of cells had undergone blast transformation, as shown by FSC and SSC profiles in FCM. In addition, entry in the cell cycle was evidenced by up-regulation of activation markers, changes in the expression pattern of CD45R0 and CD62L, and replication of genomic DNA (Fig. 1⇑C). Since CD69 is an early and transient activation marker, by day 4 in culture its expression was already declining as shown in Fig. 1⇑C. Thus, we were able to induce activation and proliferation of a large population of naive CD4+ T cells using autologous MDDC that provide important costimulatory signals.
We infected the CD4+ T lymphoblasts with HIV-1 4 days after stimulation (day 9 of the entire procedure) and followed viral replication for 15 days after infection (up to day 24 of the procedure) by intra- and extracellular p24gag Ag ELISA. As shown in Fig. 2⇓A, 10 days postinfection (day 19 of the procedure) HIV-1 p24gag Ag was detectable in the culture supernatant and inside the cells and increased markedly through day 24. Intracellular FCM analyses paralleled these results and indicated that the frequency of productively infected cells at the end of the 15-day infection period (day 24 of the procedure) was 5–10% (Fig. 2⇓B). Importantly, since the infection was conducted at a low multiplicity of infection (0.002), viral replication did not significantly affect cell viability, as shown by the high percentage of live cells in infected cultures (Fig. 2⇓A).
HIV-1 replication during the phase of cell expansion. A, Intracellular and extracellular HIV-1 p24gag levels in whole-cell extracts (•) and culture supernatants (▪), respectively. Cell viability (percentage of live cells) determined by trypan blue exclusion is also shown in this panel (⋄, uninfected cultures; ♦, infected cultures). B, Intracellular FCM analysis of HIV-1 p24gag Ag in infected and uninfected cultures. All FCM plots are gated in the live cell population as determined by FSC and SSC profiles and exclusion of 7AAD. The days of analysis reported in both panels correspond to the time scale reported in Fig. 1⇑A. Results are representative of 10 different donors.
Next, we sought to generate resting cells from HIV-infected, CD4+ T cell cultures. At day 24 of the procedure, we enriched activated (memory-phenotype) CD4+ T cells by negative selection to eliminate naive cells unresponsive to SEB (Fig. 3⇓A). Expression of CD25 and Ki-67 and genomic DNA content >2 N indicated that the enriched cell population comprised activated cells still progressing through the cell cycle (Fig. 3⇓A). Then, enriched cells were cultured with IL-7 and allowed to rest for up to 4 wk (up to day 53 of the procedure). Pilot experiments (data not shown) indicated that IL-7 at the dose used in our system did not induce proliferation compared with untreated cultures. However, IL-7 had a protective effect against cell death caused by growth factor withdrawal and promoted a faster return to quiescence, consistent with previous reports (12, 13). Resting phases shorter than 2 wk achieved cell quiescence as determined by lack of proliferation and most activation markers, although CD25 required 4 wk to disappear completely. However, short rest phases did not achieve complete suppression of viral replication (data not shown). Throughout the phase of clonal contraction, we monitored forward and side light scatter profiles, expression of activation markers, and genomic DNA content. All of these parameters indicated that at the end of the resting phase, cells had returned to quiescence (Fig. 3⇓B). After a typical 21- to 28-day resting phase, the yield of viable cells varied from donor to donor, averaging 20% of the starting number (range, 5–30%, n = 6; data not shown). Interestingly, Fig. 3⇓A shows that the memory-phenotype cells isolated by negative selection at the end of the expansion phase (day 24 of the procedure) comprised both CD62L+ and CD62L− cells. On the contrary, at the end of the resting phase (day 53 of the procedure), all cells were CD62L+ and expressed the lymphoid tissue homing marker CCR7 (Fig. 3⇓B). Previous studies have shown that CD45R0+CD62L+CCR7+ cells belong predominantly to the long-lived central memory T (TCM) cell subset (21). Thus, in our system CD62L− cells die and disappear in the course of the resting phase, whereas cells with a phenotype similar to long-lived TCM cells survive and return to quiescence. It has also been reported that IL-7-treated resting cells display a larger size than untreated resting cells (22). Indeed, we found that resting CD4+ T cells cultured in the presence of IL-7 showed larger size and increased granularity than freshly isolated naive CD4+ T cells (average FSC-H value: 397 ± 19.5 vs 276 ± 37.4; average SSC-H value: 111 ± 10.7 vs 72 ± 10.2, respectively; n = 11, p < 0.001 for both parameters).
Enrichment of activated (memory-phenotype) CD4+ T cells and generation of resting cells. A, FCM analysis of activated (memory-phenotype) CD4+ T cells enriched by negative selection from CD4+ T cells and MDDC cultures. B, FCM analysis of rested CD4+ T cells. All plots (except for FSC-H vs SSC-H plots) are gated in the live cell population as determined by FSC and SSC profiles and exclusion of 7AAD. The days of analysis reported in both panels correspond to the time scale reported in Fig. 1⇑A. Results are representative of more than 10 different donors.
We also monitored viral replication during the resting phase by assaying intra- and extracellular levels of HIV-1 p24gag Ag. Intracellular p24gag levels normalized by cell number decreased during the course of the resting phase (Fig. 4⇓A). Moreover, we did not detect p24gag accumulation in the culture supernatant after the first 7 days of resting (day 31 of the procedure; Fig. 4⇓A). In independent experiments conducted with cells of three other volunteers, we found similar intracellular p24gag levels in resting cultures containing the protein synthesis inhibitor CHX (10 μg/ml), the nucleoside analog reverse transcriptase inhibitor AZT (1 μM) and in untreated cultures (Fig. 4⇓B; ▴, □, and •, respectively). These results demonstrate that intracellular p24gag entirely reflects protein synthesized during the expansion phase and undergoing slow degradation during the resting phase, rather than the product of low-level viral replication. The decline in intracellular p24gag detected by ELISA was consistent with intracellular FCM analysis showing a decline in HIV-1 p24gag-positive cells (Fig. 4⇓C). These results demonstrate the absence of active viral replication in these cells and indicate that the HIV-1 Ags detectable inside the cells were synthesized during the expansion phase and were undergoing degradation during the resting phase. In summary, these results indicate that we were able to induce cell quiescence and viral latency, obtaining resting cells in a state of nonproductive infection.
Suppression of HIV-1 replication during the resting phase. A, Intracellular and extracellular HIV-1 p24gag levels in whole-cell extracts (•) and culture supernatants (▪), respectively. The • and ▪ at day 24 refer to the intracellular and extracellular p24gag levels measured immediately before enrichment of activated (memory-phenotype) CD4+ T cells (see also Fig. 3⇑A). B, Effect of CHX and AZT on the intracellular levels of p24gag Ag during the resting phase. ▴, CHX-treated cultures; □, AZT-treated cultures; •, untreated cultures. C, Intracellular FCM analysis of HIV-1 p24gag Ag in infected and uninfected cultures. All FCM plots are gated in the live cell population as determined by FSC and SSC profiles and exclusion of 7AAD. The days of analysis reported in all panels correspond to the time scale reported in Fig. 1⇑A. Results are representative of 5–10 different donors.
Finally, we tested whether HIV-1-infected cultures that had been rested for up to 3–4 wk would resume HIV-1 replication following secondary stimulation. As indicated above, at the end of the resting phase, the yield of viable cells averaged 20% and ranged from 5 to 30%. High frequency of dead cells may represent a confounding factor in analyses performed during secondary stimulation. Thus, at the end of the resting phase (day 53 of the procedure), we eliminated dead cells from both HIV-1-infected and control cultures by Ficoll centrifugation, yielding cell populations with >98% viability as determined by trypan blue exclusion (data not shown). These cells were then cultured for 15 days (days 53–68 of the procedure) with MDDC and SEB as described above or in the presence of immobilized anti-CD3 and anti-CD28 Abs. Fig. 5⇓A shows that HIV-1 replication, as monitored by p24gag Ag ELISA, resumed as a consequence of cell restimulation.
HIV-1 replication and cell viability during secondary stimulation with MDDC and anti-CD3/CD28 Abs. A, HIV-1 p24gag levels in whole-cell extracts (circles and triangles) and culture supernatants (squares and diamonds), respectively. B, Cell viability in HIV-1 infected (○ and ▵) and control uninfected cultures (□ and ⋄) are shown as percentage of live cells determined by trypan blue exclusion. In both panels, □ and ○ represent samples stimulated with MDDC and ⋄ and ▵ represent samples stimulated with anti-CD3/CD28. The days of analysis shown in all panels correspond to the time scale reported in Fig. 1⇑A. Results are representative of five different donors.
Since HIV-1 replication did not affect cell viability during primary stimulation (see Fig. 3⇑A), we sought to determine whether reactivation of HIV-1 replication from nonproductively infected cells during secondary stimulation affected the rate of CD4+ T cell death. As shown in Fig. 5⇑B, cell viability in HIV-1- infected cultures declined more sharply than in the uninfected cultures following secondary stimulation with either MDDC or immobilized anti-CD3/CD28 Abs. Thus, these data show that replication competent virus can be rescued from nonproductively infected cells in the course of secondary stimulation. During this process, the rate of cell death in infected cultures markedly exceeds that observed in uninfected cultures, suggesting that this in vitro model may also be suitable to investigate the mechanisms of HIV-1-driven T cell death, both direct (in infected cells) and indirect (in uninfected, bystander cells).
The FCM data in Fig. 4⇑C indicate that a small proportion of resting cells retains detectable intracellular HIV-1 p24gag throughout the resting phase. This observation raises the possibility that reactivation of HIV-1 replication during secondary stimulation (as shown in Fig. 5⇑A) derives only from that small percentage of cells that remains positive for HIV-1 p24gag even after a prolonged resting phase and not from cells that have lost expression of viral Ags while carrying the HIV-1 provirus. To address this question, we conducted the secondary stimulation in the presence or absence of AZT. In the experiment shown in Fig. 6⇓, which was conducted with cells of a donor different from the one shown throughout this study, but under the same culture conditions, the fraction of cells positive for HIV-1 as detected by FCM was ∼2% at the end of the expansion phase (Fig. 6⇓A, upper plot). Following the resting phase, the percentage of p24gag-positive cells decreased to background levels (<0.1%; Fig. 6⇓A, lower plot). When these cells were subjected to secondary stimulation with Ag-loaded MDDC, we observed a progressive increase in p24gag-positive cells (Fig. 6⇓B, top row). This was paralleled by an accumulation of p24gag Ag in the culture supernatant (Fig. 6⇓C, •). Cultures reactivated in the presence of AZT initially showed a percentage of HIV-1 p24gag-positive cells similar to that observed at the end of the expansion phase (compare the upper plot in Fig. 6⇓A with the first plot in the lower row of Fig. 6⇓B). However, at subsequent time points, cultures containing AZT showed a percentage of p24gag-positive cells close to background levels (∼0.2%). At the same time, we did not observe any accumulation of p24gag Ag in the supernatant of cultures containing AZT (Fig. 6⇓C, ▪). Thus, reactivation of HIV-1 replication during secondary stimulation derives from HIV-1-infected cells, regardless of the residual presence inside the cells of viral Ags. Moreover, the presence of antiviral drugs does not affect initial reactivation of latent provirus. Indeed, the percentage of HIV-1-positive cells observed at day 48 of the procedure closely approximates that observed at the end of the expansion phase. The complete disappearance of p24gag-positive cells at subsequent time points suggests that, although existing HIV-1-infected cells die as a consequence of cellular and viral reactivation, the presence of AZT prevents new productive infections to occur, yielding a net decline in p24gag-positive cells.
Analysis of HIV-1 replication during secondary stimulation. A, Intracellular FCM analysis of HIV-1 p24gag Ags at the end of the expansion and resting phases (upper and lower plots, respectively). B, Intracellular FCM analysis of HIV-1 p24gag Ag during secondary stimulation with Ag-loaded MDDC in the absence and presence of 1 μM AZT (plots in the upper and lower rows, respectively). C, HIV-1 p24gag levels in culture supernatants during secondary stimulation with Ag-loaded MDDC in the absence or presence of 1 μM AZT (• and ▪, respectively). All FCM plots are gated in the live cell population as determined by FSC and SSC profiles and exclusion of 7AAD. The days of analysis reported in all panels correspond to the time scale reported in Fig. 1⇑A.
We developed a culture system to recapitulate in vitro the establishment and reactivation of HIV-1 latency. Our model relies on primary human CD4+ T cells activated in response to stimulation with MDDC and infected in vitro with HIV-1. The infected cultures are then returned to a quiescent status by removal of Ag and addition of IL-7. Although high doses of IL-7 were shown to rescue HIV-1 from resting CD4+ T cells of patients under HAART therapy (23), in the present study we used low doses of IL-7 to counter cell death due to withdrawal of growth factors without inducing cell activation and proliferation or viral replication (12, 13). We demonstrated that quiescent, nonproductively infected cells obtained with this procedure lack cell surface activation markers and cell cycle markers, do not synthesize DNA, do not proliferate, and do not replicate HIV-1. Indeed, we demonstrated that p24gag Ag within infected cells represents protein expressed during the expansion phase and undergoing slow degradation during the resting phase without any de novo production. This is consistent with previous reports showing that resting cells present a highly nonpermissive environment for viral replication due to lack of critical transcription factors that require cell activation for proper function (24, 25, 26). Two recent studies presented evidence in support of the high stability of p24gag both in vivo and in vitro (27, 28). In our study, we also showed that only CD45R0+CD62L+CCR7+ cells, which in vivo represent predominantly long-lived TCM cells (21), survive through the resting phase and return to quiescence. In contrast, CD45R0+CD62L−CCR7+ cells, that are phenotypically similar to short-lived effector memory cells (21), disappear. All parameters of cell activation and proliferation and viral replication resumed following restimulation with either MDDC or immobilized Abs directed against CD3 and CD28. We also found that during secondary stimulation CD4+ T cell viability declined more sharply in HIV-1-infected than in uninfected cultures, although no significant difference was observed during primary stimulation. Current studies are aimed at assessing whether higher rates of cell death within infected cultures affect HIV-1-positive cells or uninfected, bystander cells, as well as at addressing the mechanisms underlying these phenomena. Finally, we demonstrated that reactivation of HIV-1 replication during secondary stimulation occurs from HIV-1-infected CD4+ T cells, regardless of whether p24gag Ag is still detectable in their cytoplasm at the end of the resting phase.
Early in situ hybridization studies showed that cells harboring the HIV-1 provirus were found at higher frequency than cells expressing HIV-1 mRNA, providing indirect evidence of HIV-1 latency (2). However, only in the last decade Siliciano and colleagues (3, 4) demonstrated conclusively the existence of a pool of latently infected cells by showing that unstimulated, resting CD4+ T cells from HIV-1 patients did not replicate HIV-1 in vitro, but carried integrated provirus, which could be rescued after cell activation (5).
Viral latency is the consequence of HIV-1 tropism for CD4+ T cells and the cellular mechanisms that establish immunological memory. Although viral latency does not affect the natural history of HIV-1 infection, it plays a critical role in clinical practice. Indeed, a pool of latently infected cells persists even under successful antiretroviral therapy (6, 7), with a decay rate of 44 mo that guarantees lifetime persistence of the virus (7). Moreover, the latent reservoir archives drug-resistant viruses that emerge in the context of inadequate therapy or poor compliance (29). Thus, latency influences therapeutic strategies (30) and represents a major obstacle to virus eradication.
Margolis and colleagues (31, 32, 33) demonstrated the role of the human factors YY1 and LSF in suppressing viral transcription and establishing latency through recruitment of HDAC1 to the HIV-1 promoter. However, the mechanisms involved in the establishment and reactivation of latency remain to be elucidated, primarily for the lack of a suitable cellular system. The very low frequency of latently infected cells in clinical samples and the lack of known distinctive markers represent major drawbacks for this approach (8). Moreover, clinical samples are limited to the study of reactivation of latent provirus, which can be difficult, and different approaches are still under evaluation (3, 34, 35, 36, 37, 38). Several laboratories have established in vitro models of HIV-1 latency involving the use of chronically infected cell lines with minimal constitutive but inducible HIV-1 gene expression (e.g., ACH2, U1, JΔK) and clonal cell lines derived after transduction with engineered viral vectors (e.g., J-Lat; Refs. 39, 40, 41, 42). Although these models afford a reliable supply of latently infected cells to investigate both establishment and reactivation of latency, they lack the physiological relevance of systems based on primary cells. Under conditions of constitutive cellular activation and proliferation, latency may be the consequence of proviral integration within heterochromatic regions of the host genome (42), whereas in vivo HIV-1 integrates primarily within transcription-active regions (43). This suggests that different mechanisms of latency may be called into play under cellular and genomic environments so diverse. Thus, an ideal model should comply with both the requirement of viral latency and cellular quiescence, which is best afforded by systems that rely on primary cells.
Recent studies reported the generation of latently infected cells through infection of resting CD4+ T cells that were either unstimulated or that had been pretreated with the CCR7 ligands CCL19 and CCL21 (44, 45). In that report, stimulation with CCL19 and 21 did not induce expression of activation markers and did not promote cell proliferation, but led to high rates of proviral integration despite low levels of viral replication. Upon cell stimulation, a marked increase in viral production was detected (45). HIV-1 infection of resting cells in vivo is characterized by entry and reverse transcription, but not integration and production of viral progeny; thus leading to a status termed preintegration latency (46, 47). Under these circumstances, the unintegrated provirus is labile and rapidly degraded (half-life of ∼2 days), suggesting that infection of resting CD4+ T cells is unlikely to play a role in long-term viral persistence (48).
In the system reported by Hirsh and colleagues (49), resting cells were generated after depletion of residual activated cells 3 wk following PHA activation of CD4+ T cells infected in vitro with HIV-1. These cells displayed persistent viral production, possibly the consequence of continued addition of IL-2 in the culture medium to prevent cell death (49). Indeed, although these cells lacked cell surface expression of CD25, engagement of the intermediate-affinity IL-2R (formed by the β- and common γ- chains, CD122 and CD132) triggers intracellular signals that support cell activation and proliferation (50). In addition, monitoring of viral replication was limited to the first 4–7 days, while a longer resting phase might have shown waning viral replication.
The in vitro model of HIV-1 latency we described here presents several advantages: 1) use of primary cells, 2) culture methods that recapitulate a wide array of stimuli at play in vivo, 3) compliance with both viral latency and cellular quiescence requirements, and 4) suitability to study mechanisms at play during both establishment and reactivation of latency. In addition, the observation of higher cell death rates within HIV-1-infected than uninfected cultures during secondary stimulation indicates that this culture system may provide a model to study the mechanisms of infected and uninfected bystander CD4+ T cell depletion following HIV-1 reactivation.
Resting, latently infected cells present in vivo and in clinical samples have the distinctive feature of being true long-lived memory cells that cannot be faithfully generated in vitro with current technology. However, the quiescent, nonproductively infected cells we described here represent an advancement from current in vitro models and a better approximation of the in vivo situation, which may prove extremely valuable in advancing our understanding of HIV-1 latency.
We thank Drs. Davide Zella, George K. Lewis, and Yongjun Guan for helpful discussions and Dr. Clara Lehmann for critical reading of this manuscript. We are especially grateful to Dr. Robert C. Gallo for continued support.
↵2 Abbreviations used in this paper: HAART, highly active antiretroviral therapy; MDCC, monocyte-derived dendritic cell; 7AAD, 7-aminoactinomycin D; SEB, staphylococcal enterotoxin B; CHX, cycloheximide; AZT, azidothymidine; FCM, flow cytometry; TCM, central memory T; FSC-H, forward scatter height; SSC-H, side light scatter height.
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Alcamí, J., T. Laín de Lera, L. Folgueira, M. A. Pedraza, J. M. Jacqué, F. Bachelerie, A. R. Noriega, R. T. Hay, D. Harrich, R. B. Gaynor, et al 1995. Absolute dependence on κB responsive elements for initiation and Tat-mediated amplification of HIV transcription in blood CD4 T lymphocytes. EMBO J. 14: 1552-1560.
Pendergrast, P. S., N. Hernandez. 1997. RNA-targeted activators, but not DNA-targeted activators, repress the synthesis of short transcripts at the human immunodeficiency virus type 1 long terminal repeat. J. Virol. 71: 910-917.
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Brussel, A., P. Sonigo. 2004. Evidence for gene expression by unintegrated human immunodeficiency virus type 1 DNA species. J. Virol. 78: 11263-11271.
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Yeni, P. G., S. M. Hammer, C. C. Carpenter, D. A. Cooper, M. A. Fischl, J. M. Gatell, B. G. Gazzard, M. S. Hirsch, D. M. Jacobsen, D. A. Katzenstein, et al 2002. Antiretroviral treatment for adult HIV infection in 2002: updated recommendations of the International AIDS Society-USA Panel. J. Am. Med. Assoc. 288: 222-235.
Romerio, F., M. N. Gabriel, D. M. Margolis. 1997. Repression of human immunodeficiency virus type 1 through the novel cooperation of human factors YY1 and LSF. J. Virol. 71: 9375-9382.
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He, G., D. M. Margolis. 2002. Counterregulation of chromatin deacetylation and histone deacetylase occupancy at the integrated promoter of human immunodeficiency virus type 1 (HIV-1) by the HIV-1 repressor YY1 and HIV-1 activator Tat. Mol. Cell. Biol. 22: 2965-2973.
Chun, T. W., D. Engel, S. B. Mizell, L. A. Ehler, A. S. Fauci. 1998. Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines. J. Exp. Med. 188: 83-91.
Moriuchi, H., M. Moriuchi, A. S. Fauci. 1999. Induction of HIV-1 replication by allogeneic stimulation. J. Immunol. 162: 7543-7548.
Korin, Y. D., D. G. Brooks, S. Brown, A. Korotzer, J. A. Zack. 2002. Effects of prostratin on T-cell activation and human immunodeficiency virus latency. J. Virol. 76: 8118-8123.
Brooks, D. G., D. H. Hamer, P. A. Arlen, L. Gao, G. Bristol, C. M. Kitchen, E. A. Berger, J. A. Zack. 2003. Molecular characterization, reactivation, and depletion of latent HIV. Immunity 19: 413-423.
Shen, A., H. C. Yang, Y. Zhou, A. J. Chase, J. D. Boyer, H. Zhang, J. B. Margolick, M. C. Zink, J. E. Clements, R. F. Siliciano. 2007. Novel pathway for induction of latent virus from resting CD4+ T cells in the simian immunodeficiency virus/macaque model of human immunodeficiency virus type 1 latency. J. Virol. 81: 1660-1670.
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UA System approves tighter budgets – Northwest Arkansas Democrat-Gazette
University of Arkansas System trustees Thursday approved reduced budgets at most campuses for next year, the result of cautious planning for lower state revenue and shrinking enrollment.
After years of increasing tuition and fees to cover rising operating costs, system schools are keeping those rates the same while instituting or continuing hiring freezes and reducing spending on supplies and services. Most have said they aren't currently planning layoffs.
Trustees approved the budgets on a voice vote without opposition. Collectively, they trim tens of millions of dollars from the schools' budgets.
Colleges nationwide are grappling with financial losses and dismal enrollment and financial projections for the fall. Many public institutions have already struggled with declining state revenue since the Great Recession.
In Arkansas, state revenue had largely been stagnant, rather than shrinking, until the coronavirus pandemic. Several schools have created committees to look at the pandemic's impact and explore what other budget reduction options they may have in the near future, including layoffs.
University of Arkansas System budgets, which contracted at most schools and expanded slightly at others, were submitted by the chancellors of each campus, but not without direction from system administrators.
Trustees directed the chancellors to keep tuition and fees flat for the fall while reserving the right to increase them, if needed, for the spring. System President Donald Bobbitt asked the chancellors to create their budgets assuming that state revenue would cover only certain general appropriation categories, while still fulfilling most of what has been promised to campuses.
Gina Terry, system vice president of finance and administration, said planning for next year's budgets was the most challenging effort of her career.
"Just the uncertainties that are out there right now with housing and enrollment and athletics," she said. "It really challenges each campus to try to figure out how to manage and try not to have layoffs, if they can help it."
The University of Arkansas at Monticello reported plans to lay off at least one person. Details on the number of people laid off and what their jobs were have been unavailable this week. The university budgeted expenditures of $3.3 million less next year compared with what was approved for this year.
The approved spending decreases at the system's schools, compared with last year's approved expenditures, represent as much as 11.7% less.
That percentage is the amount stripped from the budget for the University of Arkansas at Pine Bluff, the state's only public historically black college or university.
At the flagship institution, the University of Arkansas, Fayetteville, cuts amount to just more than 2% of last year's approved expenditures.
Out of the system's five traditional four-year institutions, its medical school and its seven community colleges, all but four expect a deficit next year, to be covered by reserves. UALR expects to spend $9.9 million in reserves to cover its shortfall, and the University of Arkansas at Fort Smith intends to spend $6.5 million of its $9 million in reserves.
The traditional four-year universities and seven community colleges collectively axed $41.5 million from their proposed expenditures, compared with what was approved last year. That's out of $1.3 billion in planned expenses, or a 3.2% spending decrease.
Universities were hit the hardest, while the largest budget reduction at a community college was 3.8% at the University of Arkansas Community College at Hope-Texarkana.
There, administrators reduced compensation by planning to use adjunct faculty and part-time help, a common tactic when colleges cut expenses. The college is also deferring about $250,000 in critical maintenance.
The college expects to lose 20% of its typical local sales tax revenue and 20% of its typical funding from Workforce 2000, part of its state appropriation.
Trustee Cliff Gibson thanked the schools for looking as deeply inward as they did to find cuts.
"What anecdotally I've learned is that everybody was pulled in, right down to the guy that mows the yard, trying to figure out, 'What can we do?'" he said.
UA System President Donald Bobbitt said years of conservative budgeting and prioritizing healthy "rainy day" reserve funds at each campus has helped keep the impacts minimal.
"And it's rainy," he said.
Projections for fiscal 2021 compared with fiscal 2020 approved budgets
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Erin and Ben Napier of "Home Town" have done some massive makeovers, but these HGTV stars also know that not every homeowner can afford...

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The Sonepar Group is multinational group based in France which has quietly assembled a network of companies. When I accessed the "about-us" page on Sonepar USA, the page claimed that, in 2015, Sonepar had $22.8 Billion in sales from over 2,800 branches with 43,000 associates.
The company was founded by Henrí Coisne in 1969. It did not jump the Atlantic until 1984 when it acquired a Quebec based company caled Lumen.
US acquisitions increased after Sonepar hired Richard Worthy from GE Capital in 1998. Worthy engaged in a disciplined acquisition of electronic contractors and suppliers and developed new branches when they fit.
The company uses a distributed management style. When it acquires companies, it keeps the name and leaves the management structure in place. This page will list companies in the Sonepar USA division.
Brook Electrical Supply lists 5 offices in Illinois. The company was founded in 1927 and acquired in April of 2000.
Capital TriState lists 54 offices in DC, DE, MD, PA, VA, WV. They obviously don't know what "Tri" means.
Coldale Electic Supply was founded in 1975 by Dale P. Holt and was acquired by Sonepar in 2012. Their locations page listed 15 stores in Utah, Nevada and Wyoming.
Copper Electric serves NJ, NY and Pa. They list 29 offices.
Crawford serves Louisiana and Texas. They list 24 offices.
Eck serves both North and South Carolina. It has seven offices.
Eck was founded in 1955. Sonpar acquired the company in 2015.
Electrical Distributors was founded in 2008. It has two locations in Texas.
Eoff serves Washington and Oregon. It was founded in 1919 in Salem, Oregon. The site lists 11 locations.
Friedman was founded in 1935 and acquired in 2006. They list 15 locations in New York and Pennsylvania.
Independent Electric Supply was founded in 1976 and acquired by Sonepar in 2011. They list 18 locations in Arizona and California.
Irby was founded in 1926. Irby was the 16th largest electrical dealer with 35 stores and $400M in sales when in was acquired in October 2005. The site listed 62 locations in AL, AR, CO, FL, GA, IL, KY, LA, ME, MA, MI, MN, MS, MO, NE, NJ, NM, NY, ND, OK, SD, TN, TX, VA, WI in January 2018.
NorthEast Electrical was created in 1998. Its site listed 31 locations in CT, MA, ME, NH, RI, and VT.
OneSource Distributors lists 20 offices in Arizona, California and Hawaii. It was created in 1983 as San Diego Wholesale Electric. It changed its name to OneSource in 1996 and was acquired by Sonepar in 2011.
QED (Quality Electrical Distribution) was founded in Denver in 1987. Their site listed 10 offices in Arizona, Colorado, North Dakota, Nevada and Utah.
V Allen was itself a conglomerate before acquisition. Their map page listed 68 US locations in AL, AZ, AR, CA, CO, CT, FL, GA, KS, KY, LA, MI, MN, MO, NH, NY, NC, ND, OH, PA, SC, SD, TN, TX, WA, WV, and WI.
In a complex series of acquisitions in 2007-2008, Sonepar acquired a conglomerate called IDG Group and a portion of a company called Hagemeyer with a competitor named Rexel acquiring the bulk of Hagemeyer. Sonepar had a branding problem and finally branded the division as Vallen.
The IDG Group was created from 1997-1999 with the consolidation of 31 companies including 9 major distributors. The oldest and most prestigious of these was the Cameron & Barkley Company that was founded in Charleston, SC in 1865.
According to Wikipedia (drawn 1/2018), The company called Hagemeyer was created in Munich in 1905. In 2007 Sonepar and Rexel competed for the acquisition. Rexel won but sold some of the pieces to Sonepar.
Viking was founded in 1964 in St Paul. They list 21 locations in Minnesota and Wisconsin.
World Electric Supply was founded in 1999. It has ten locations in Florida.


Description: This lab exercise demonstrates the usage of the portfast command on the switch.
1. Enter into the global configuration mode on the switch.
2. Use the command "Spanning-tree portfast" to enable portfast feature on the switch and verify the same using show command.
3. Use the no form of the above command to disable the portfast on switch and verify the same using show spanning-tree summary command.


text:
With Proposed Bill, America Publicly Embraces the Age of Hypersonic Warfare
Sam Mire, June 14, 2018 11:30AM
Allow me to quote briefly the rock supergroup of our generation, Imagine Dragons.
"Welcome to the new age, to the new age
Whoa, oh, oh, oh, oh, whoa, oh, oh, oh"
Recent bipartisan legislation introduced in the Senate confirms that truer lyrics have not been penned.
We are in a new age; the age of open, unabashed hypersonic armament.
How quickly the future sneaks up on us.
Despite a military budget significantly depleted in recent years, the current administration has proposed increases in military spending by as much as $85 billion. Fortification of America's arsenal has been underway virtually since Donald Trump's inauguration – it is one of his highest, most publicly avowed priorities.
And, with Vladimir Putin hard at work logging distance between Russia's military technology and America's, which has been hamstrung by unrealistic budget cuts and misallocated spending, the world's foremost superpower has some catching up to do. Putin's unveiling of an air-launched hypersonic weapon – what appears to be a modified Iskander ballistic missile – just over three months ago has undoubtedly applied pressure on American leadership to respond in kind.
Kickstarting that response is precisely what U.S. Senators Brian Schatz (D-Hawai'i) and Dan Sullivan (R-Alaska) aim to do with their introduction of Senate Bill 2980, also known as the Integrated Missile Defense Act of 2018.
"Last year, Congress – working closely with the Trump administration – undertook much-needed efforts to dramatically bolster and advance our country's missile defense," said Senator Sullivan. "This year, continuing to work in a bipartisan fashion, our bill finally authorizes the full development and deployment of a space-based sensor layer." (schatz.senate.gov)
The bill follows the passage of the Advancing America's Missile Defense Act of 2017 last year. While the military is constantly developing technologies that the public, and American politicians, aren't aware of, funding for and contracting of massive projects typically requires Congressional approval.
The bill was announced before in the days before the Trump-Kim Singapore Summit, and justification for strengthening American missile defenses includes deterrence of any threats levied by the Kim regime.
"When it comes to North Korea, we can hope for the best while still planning for the worst," Senator Schatz said. "I strongly support diplomacy, but in the meantime, this bill beefs up our missile defense system and protects Hawai'i, Alaska, and the U.S. mainland from rogue missile threats."
But, ultimately, the development of hypersonic weaponry is necessary if America is to reclaim its position as leader in military technology development. Leadership aside, such weapons are needed, if only to keep up with Russia's defenses.
"[Hypersonic missile defense] also speeds up our efforts to protect U.S. forces and allies in the region by improving our ability to detect, track, discriminate, and intercept increasingly sophisticated future missile threats," Schatz added.
The details of what, exactly, this bill would aim to do paint a picture of the new age of space-based defensive armaments where the technologies formerly of the future now qualify as present threats. The bill:
Develops and Deploys Space-based Sensors: Mandates the development deployment of space-based sensors as soon as practicable.
Readies Our Defenses: Mandates an analysis of accelerating the development and deployment of the Redesigned Kill Vehicle (RKV) to Missile Field 4 at Fort Greely.
Promotes a More Integrated Missile Defense: Directs a study on an integrated air-and-missile defense architecture to protect against evolving threats outlined in the 2018 National Defense Strategy.
Accelerates Our Defenses Against Hypersonic Threats: Directs the acceleration of our hypersonic missile defenses and links them to the deployment of space-based sensors.
Focuses of Allies: Expresses that the U.S. should work with allies and trusted partners to share missile defense capabilities.
More Rigorous Testing: Seeks to discourage a risk adverse culture of missile defense testing and promotes a more rigorous testing regime to deliver capabilities at the "speed of relevance." (schatz.senate.gov)
Policies of military deterrence have always precipitated arms races, and with those races inevitably comes the development of newer, more potentially destructive technologies. The frightening nature of a given military technology – in this case, hypersonic ballistic missiles – can only be allayed by the fact that adversaries and their foes each possess them, enacting the tenuous safety net that is mutually assured destruction.
The development of these weapons falls within the parameters of the Army's Modernization Strategy, which calls for improving 'Long-Range Precision Fires' and 'Air and Missile Defense Capabilities'. Now, it's been announced that the hypersonic missile project, dubbed "Hacksaw", will be completed under the watch of trusty contract Lockheed Martin.
'The U.S. Air Force awarded major defense contractor Lockheed Martin a $928 million contract to develop a weapon designed to fly more than five times faster the speed of sound. These hypersonic weapons are designed to overwhelm enemy defenses with blistering speed and provide the capability to strike targets where the time window is tight.' (Yahoo via Air Force Magazine)
Though some may see this bill and technology as hawkish, the fact that Russia has proven to possess this technology makes the bill imperative. Not because war with Russia, North Korea, or Iran is imminent or desirous – here's to the naïve hope that peace will last forever. But, because a level playing field is one of the surest-fire ways to avoid conflict in the first place.
Sam Mire writes about US politics and US foreign policy for TrigTent.


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Global Day For Decent Work: COSATU Calls A Nationwide Stay-Away On 7 October 2020
The Congress of South Africa Trade Unions (COSATU) called for a nationwide stay away on 7 October 2020, in commemoration of the 13th Global Day for Decent Work and the ongoing plight of workers as a result of the COVID-19 pandemic.
The Global Day for Decent Work is celebrated annually by trade unions worldwide as a call to governments to take action in relation to economic challenges faced by workers and to compel them to provide "decent work" as a mechanism to achieve economic growth and a people-centred economy. In its September 2020 media statement, COSATU noted that: "South Africa is teetering on the brink of collapse and it is about time we all stand up and demand urgent action from policymakers and decision-makers".
So, what are the issues being raised by COSATU? They include the following:
1. a lack of Personal Protective Equipment for frontline workers;
2. an emphasis on the failure of the public transport system;
3. the undermining of collective bargaining in the public service by the state;
4. the general inefficiency of the state;
5. the scourge of corruption in South Africa;
6. the loss of some ZAR80 billion annually through transfer pricing manipulation;
7. the looting of the COVID-19 UIF TERS Fund by employers;
8. mismanaged and now cash strapped or bankrupt SOE's;
9. the maximum penalty threshold which can be imposed by the Competition Commission is inadequate;
10. Continued cartel conduct;
11. the amendment to the Competition Act 89 of 1998 was meant to provide for directors and managers to be criminally prosecuted, but thus far there have been no prosecutions; and
12. families of politicians should be barred from doing business with the state.
The issues raised by COSATU are broad and for the most part the majority of South Africans already support action to rid the state of inefficacies and corruption and seek to hold criminals within government and the private sector accountable. What is interesting to note is the emphasis on fraud in relation to TERS as well as the focus on commercial issues like transfer pricing and anti-competitive conduct. Like in other parts of the world, fraud perpetrated against the state in the provision of COVID-19 employee benefits has attracted civil and criminal liability.
This is also the case in South Africa. Companies which received TERS benefits are well advised to conduct proper audits on the receipt and disbursement of benefits. Also, companies should be alive to transfer pricing manipulation and anti-competitive conduct and guard against such conduct which is not only unlawful but appears, in time, that it will also be attracting a lot more public scrutiny.
Significant about the nationwide protest called by COSATU is that this constitutes protest action in terms of section 77 of the Labour Relations Act 66 of 1995 (LRA) which section affords employees the right to engage in "protest action to promote or defend socio-economic interests of workers". Section 77(1) of the LRA provides for procedural requirements that must be adhered to by trade unions when embarking on a protest in terms of the section. These requirements are: (i) the protest must be called by a registered trade union or federation of trade unions; (ii) the National Economic Development and Labour Council (NEDLAC) must be provided with a notice of the proposed protest stating the nature and purpose thereof; (iii) the matter giving rise to the proposed protest must then be considered by NEDLAC or an appropriate forum and (iv) the trade union must provide NEDLAC with a notice of its intention to proceed with the proposed protest action 14 days prior to the commencement of same.
Whilst the protest action was called for by COSATU, it is also endorsed by the Federation of Unions of South Africa (FEDUSA) and the National Council of Trade Unions (NACTU), which are all represented at NEDLAC. Breakaway federation SAFTU (the South African Federation of Trade Unions) is not part of NEDLAC, but will also participate in the 7 October 2020 stayaway. COSATU and SAFTU make strange bedfellows for a number of historic reasons.
As the protest action is authorised in terms of section 77(2) of the LRA, persons who engage in the protest action are afforded protection under the LRA, including the protection against dismissal for participating in the protest action.
The procedural requirements set out in section 77(1) of the LRA must be complied with and the courts take a dim view on trade unions who fail to do so. In the recent Labour Court judgment of Business Unity SA v Congress of South African Trade Unions & others (2020) 41 ILJ 174 (LC), a nationwide protest was called by COSATU and other trade unions against a draft Employment Standards Bill. Business Unity SA obtained an interdict in the Labour Court against the proposed protest action on the basis that the respective trade unions had not complied with the procedural requirements of the LRA. COSATU argued that the right to protest is a manifestation of the right to strike.
The Labour Court disagreed with COSATU and emphasised that there is a clear distinction between the right to strike and the right to protest action. Hence, the LRA provides for different procedural requirements for the right to protest and the right to strike. This distinction is one which finds support in international law where a differentiation is made between industrial action underpinning the collective bargaining process and a work stoppage for "political" purposes (such as the broad socio-economic interests of workers).
The practical effect of the nationwide protest action on employers is that their workforce may participate in the protest action or employees may be unable to attend work due to the non-availability of public transport.
All this at a time where many businesses have only recently recommenced operations after the national state of disaster was announced in March 2020 and which continues. Businesses are therefore in a precarious position as they cannot prevent employees from participating in the protest action but also cannot afford to have their employees away from work whilst seeking to at least recover some of the financial losses suffered as a result of the pandemic.
The law establishes that the purpose of informing NEDLAC of the proposed protest is to allow for government, labour, business and community organisations to cooperate, through problem-solving and negotiation on economic, labour and development issues as well as related challenges facing the country. Only time will tell what impact this stay away will have on the course of history, as the country must navigate through the continued uncertainty of COVID-19 as well as the already failing economy which South Africa was faced with before the pandemic, all against the backdrop of a global recession.
Does trading under the African Continental Free Trade Agreement (ACFTA), which becomes operational in January 2020, at least create a green shoot which COSATU should be focussing on like many innovative businesses? Will the ACFTA bring the necessary economic growth?
Tags: Cosatu Global Day For Decent Work
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These actresses are some of the most popular from 1970s television whose beauty and expertise have been showcased in such shows as Charlie's Angels and Dukes of Hazzard, amongst others as properly. Granted, this is not your typical science fiction film, however in protecting matters akin to biology, faith, history, archeology, anthropology, and the nature of human psychology (up to now and the present) in a sensible and sometimes accurate method, the movie finally ends up being probably the most excellent, insightful, and thought frightening blends of science and make-believe to ever hit the display screen.
Written with an nearly poetical high quality, The Woman who Sailed the Soul is a dark romance, a psychological examine, a haunting area opera, a wildly ingenious science and, ultimately, a fairy story. The new science fiction area opera novel by Peter F Hamilton 2013 – Nice North Street – an epic area opera sci fi ebook and thriller in a single. College leaders attended this Science and Know-how/Engineering Standards info session to be taught more in regards to the new draft of the PreK-12 standards which might be slated for a fall 2015 implementation.
Oftentimes documentaries about motion pictures and the film business will (unsurprisingly) reference and speak at size about motion pictures of the past that have some reference to the subject of the documentary you are watching. If asked to cite a favorite science fiction brief story by Lewis Padgett, many readers would pick the advanced and interesting Mimsy Had been the Borogoves, on which the 2007 film The Final Mimsy was based. These would usually be explained by science to be included in science fiction. They will also level you to sources you should use like books or credible websites like on-line journals.
Pay attention to the directors too, though, especially with the flicks that stick out to you essentially the most. Zones of thought is a thought frightening and great trilogy, a should read for the science fiction aficionado. Farewell to the Grasp was an important story, while in some senses it was typical of what made Golden Age science fiction great. Whereas it might lack the poetry of Contact, it's nevertheless a showcase of simply how, and how effectively, science and scientists work collectively to do amazing things. Conventions are the perfect place to meet other individuals, hear fresh concepts, and to see a mixture of science fiction fandoms that would spark an inspiration or allow you to discover a part of sci fi you really like.
Two science fiction nerds (Shaun of the Useless's Simon Pegg and Nick Frost) take a pilgrimage in an RV to America's UFO heartland, where they happen upon a smart-cracking house alien named Paul (played by Seth Rogen), who has recently escaped a 60-year imprisonment from a top-secret military base.


text:
Q: excel VBA delete rows after autofilter hello I am trying to create a button for a part of the business I work in, that filters on a Criteria, then deletes those entire rows.
I am fine with the "Filtering" part of it, and that is a success, I just cannot delete the entire rows that have been filtered. I have tried different codes and the below is the last one I tried and failed with. the failure happens beneath the 'Delete rows comment
Sub delete_stage()

Dim ws As Worksheet

Set ws = ThisWorkbook.Worksheets("Sales Pipeline Report")
ws.Activate

'Clear any existing filters
On Error Resume Next
ws.ShowAllData
On Error GoTo 0

' filter cells on criterea

ws.Range("$E$2").AutoFilter Field:=4, Criteria1:="Onboarding"

'Delete Rows

 ws.Range("$E$2").Offset(1, 0).SpecialCells(xlCellTypeVisible).EntireRow.Delete

'3. Clear Filter
On Error Resume Next
ws.ShowAllData
On Error GoTo 0

Hope someone can help. I feel it shouldn't be as hard as I am making it :-s

A: Delete Filtered Rows (AutoFilter)
The Code
Option Explicit

Sub deleteFilteredRows()

    ' Define constants.

    Const wsName As String = "Sales Pipeline Report"
    Const FirstCell As String = "E2"
    Const afField As Long = 4
    Const Criteria As String = "Onboarding"

    ' Define Source Range.

    ' Define Success Boolean.
    Dim Success As Boolean
    ' Define Worksheet.
    Dim ws As Worksheet
    Set ws = ThisWorkbook.Worksheets(wsName)
    ' Define First Cell Range ('cel').
    Dim cel As Range
    Set cel = ws.Range(FirstCell)
    ' Define Current Region ('rng').
    Dim rng As Range
    Set rng = cel.CurrentRegion
    ' Define Source Range ('rng').
    Set rng = cel _
      .Resize(rng.Rows.Count + rng.Row - cel.Row, _
              rng.Columns.Count + rng.Column - cel.Column)

    ' Delete filtered rows.

    ' Turn off screen updating.
    Application.ScreenUpdating = False
    ' Turn off AutoFilter.
    If ws.AutoFilterMode Then
        ws.AutoFilterMode = False
    End If
    ' Filter Source Range.
    rng.AutoFilter Field:=afField, Criteria1:=Criteria
    ' Try to define Filtered Range ('rng').
    Dim dRng As Range
    On Error Resume Next
    Set dRng = rng.Resize(rng.Rows.Count - 1).Offset(1) _
                                             .SpecialCells(xlCellTypeVisible)
    On Error GoTo 0
    ' Validate Filtered Range.
    If dRng Is Nothing Then
        GoTo SafeExit
    End If
    ' Delete entire rows of Filtered Range.
    dRng.EntireRow.Delete
    ' Validate Success.
    Success = True
SafeExit:
    ' Clear filter.
    ws.ShowAllData
    ' Turn on screen updating.
    Application.ScreenUpdating = True

    ' Inform user.

    If Success = True Then
        MsgBox "Rows of filtered data deleted.", vbInformation, "Success"
    Else
        MsgBox "No action taken.", vbCritical, "Fail"
    End If

End Sub

redpajama_set_name: RedPajamaStackExchange


A study of how drugs can prevent the transmission of HIV has been named Science journal's Breakthrough of the Year.
The HPTN 052 trial recruited almost 1,800 heterosexual couples from nine countries, each pair including one partner infected by the HIV virus that triggers Aids.
Anti-retroviral drugs were given immediately to half of the infected individuals.
Earlier this year the dramatic impact of the treatment led to the trial being halted four years early.
The results showed that people infected with HIV are 96 per cent less likely to pass the virus to their partners after ARV treatment.
The journal's top 10 list of groundbreaking scientific achievements from 2011 also included:The Hayabusha Mission: Japan's Hayabusa spacecraft returned to Earth with dust from the surface of a large S-type asteroid.Unravelling human origins: Researchers discovered that many people still carry DNA variants inherited from archaic humans.Capturing a photosynthetic protein: Scientists in Japan mapped the structure of plant proteins used to split water into hydrogen and oxygen atoms.Pristine gas in space: Astronomers using the Keck telescope in Hawaii detected two clouds of hydrogen gas that appear to have maintained their original chemistry for two billion years after the Big Bang.Getting to know the microbiome: Research on the countless microbes that live in the human gut showed that everyone has a dominant bacterium in their digestive tract.A promising malaria vaccine: Early results from a clinical trial that enrolled more than 15,000 children raised hopes of developing a successful malaria vaccine.Strange solar systems: Astronomers probed several distant planetary systems and came up with some bizarre findings.Designer zeolites: Zeolites are porous minerals described as 'molecular sieves' that have a broad range of applications. This year a new range of cheaper, high performing zeolites were developed.Clearing senescent cells: Research showed that clearing 'senescent' cells – cells which have stopped dividing – from the bodies of mice can delay age-related symptoms such as cataracts and muscle weakness.


Mario & Luigi Go Home 2 involves the brothers Mario teaming up to try to beat each level. It is a fun game where you must collect all the coins on the level and then reach the little castle to win. You will need to eat various fruits in order to reach various parts of each level (see instructions under game). There are 10 levels in all.


text:
Duplantis vs Kendricks in a big showdown
by Anthony Foster Monday, January 25, 2021 9:56 pm
DUSSELDORF, Germany – World record-holder Armand Duplantis will open his season at the ISTAF Indoor in Dusseldorf on 31 January.
Armand "Mondo" Duplantis Mondo
The organizers confirmed Duplantis' appearance for the ISS Dome Hall event.
Duplantis, who broke pole vault WR for 2nd time in two weeks in February 2020, will face two-time world champion Sam Kendricks, world bronze medallist Piotr Lisek from Poland along with top Germans Bo Kanda Lita Baehre and Raphael Holzdeppe.
On the track, Switzerland's Ajla Del Ponte faces leading Germans Lisa Kwayie, Tatjana Pinto, Rebekka Haase, and Jennifer Montag in the women's 60m.
German champion Deniz Almas faces Ivory Coast's Arthur Gue Cisse in the men's 60m.
Pascal Martinot-Lagarde competes in the men's 60m hurdles, while Nadine Visser takes on Belarus' Alina Talay in the women's 60m hurdles.
We want to hear from you, but before you do, HIT the share buttons below to share this and other stories with friends.
Join our lively discussion by leaving a comment below. Also, join OUR Message Boards (FORUM) for more discussions —- Please remember to follow us on social media – subscribe to our social media pages –
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TagsArmand DuplantisfeaturedSam Kendricks
Anthony Foster
Is a two-time Jamaica sports journalist of the year (2004 and 2005) and world-renowned journalist. One of his award-winning articles was on Usain Bolt, 6-time Olympic champion, 11-time World champion and sprint double record holder, was published in the Jamaica Gleaner in 2004. Anthony has covered Olympic Games in Beijing 2008, London 2012 and Rio 2016; Six (6) IAAF World Athletics Championships between 2007 and 2019 and several other international sporting events, including the 2007 World Cup of cricket and his favourite football team, Argentina vs the USA in 2004.
Jamaica beat USA in men's 4x200m at Dr. Sanders Invitational
By Vijay
Asafa Powell Narrowly Misses Sub-10 in Germany
Brown set to join Kevona Davis at Texas Longhorns


is awarded the prize/grant 2017 for his work on the development and characterization of physiological hepatic model systems with the goal to study the molecular basis underlying inter-individual differences in drug response.
Volker M. Lauschke received his Ph.D. from the EMBL and the Combined Faculty of Natural Sciences and Mathematics of the University of Heidelberg in 2013 for his work on tissue patterning using molecular oscillators. In 2014, he moved to Karolinska Institutet (KI) as a MarieCurie fellow to work on the development and characterization of physiological hepatic model systems with the goal to study the molecular basis underlying inter-individual differences in drug response. After 2 years of postdoctoral studies at KI, Volker became Assistant Professor in Liver Function and Regeneration at the Department of Physiology and Pharmacology in 2017.
How a single cell develops into a fully functional organism is one of the central questions in biology. During development, patterning of the embryo must be tightly coordinated to changes in its overall size. This phenomenon of maintaining proportionality between developing structures, termed scaling, requires that cells have information about their relative position within a tissue.
In my doctoral studies I developed a primary cell culture assay in which scaling could be observed in an experimentally accessible in vitro context. Using this platform I found that spatial information can be encoded using two molecular oscillators with distinct kinetics. One oscillator is phase-shifted between neighboring cells resulting in gene activity waves sweeping across the tissue; the other is a globally synchronized reference oscillator. A famous analogy to this concept is the solution to the longstanding problem of longitude measurements in navigation. Here, the phase-shift between two clocks, one reference clock set to Greenwich time and one set to local time, was used to decode spatial, i.e. longitude coordinates.
These findings, published in Nature and distinguished as a Research Highlight in Nature Genetics and with a dedicated News and Views report in Nature, prompted a change in perspective as they revealed a conceptually distinct mechanism for positional information and scaling that utilizes phase-shifted oscillators to directly and naturally encode spatial cues in a biological system.
The liver has a unique regenerative capacity that is tightly linked to the capability of hepatocytes to rapidly respond to various signaling molecules or changes in environment. Whereas hepatocytes are functionally highly specialized and do not divide under homeostatic conditions, upon liver damage, they transiently lose expression of key hepatic genes and dedifferentiate into fetal-like progenitor cells, which enter the cell cycle to regenerate the damaged tissue. Once cells sufficiently proliferated, they redifferentiate again into mature, fully functional hepatocytes.
In my postdoctoral studies as a Marie Curie fellow, I have applied my knowledge in ex vivo primary cell culture systems and cellular signaling to contribute to the development of a 3D spheroid culture system for primary human hepatocytes in which cells remain viable and functionally stable for multiple weeks. Strikingly, in this system cells first lose their hepatic characteristics during the initial aggregation phase but regain them after spheroids formed, providing an ideal ex vivo experimental paradigm to study the full spectrum of differentiation state changes that occur in vivo during liver regeneration.
My research group tries to elucidate the mechanistic underpinnings that grant hepatocytes their unique plasticity. To this end, we employ a multidisciplinary approach combining integrative multi-dimensional omics analyses for hypothesis generation with targeted experiments in vitro and in vivo to functionally test interesting findings. In addition, we aspire to utilize the obtained knowledge to develop approaches to generate functional hepatocytes in vitro that can be utilized for autologous transplantation purposes.
I am deeply grateful to have been granted the Malin och Lennart Philipson Award 2017. During my predoctoral studies I had the pleasure to experience the inclusive, approachable and open-minded spirit that he managed to evoke at EMBL and which coined so many scientists throughout Europe and worldwide, including myself. I feel honored to receive a prize in Lennart's name and will strive to contribute to the dissemination of his scientific leadership philosophy.


text:
package org.hibernate.envers.test.integration.modifiedflags;

import java.util.List;
import javax.persistence.EntityManager;

import org.junit.Test;

import org.hibernate.ejb.Ejb3Configuration;
import org.hibernate.envers.query.AuditEntity;
import org.hibernate.envers.test.Priority;
import org.hibernate.envers.test.integration.auditReader.AuditedTestEntity;
import org.hibernate.envers.test.integration.auditReader.NotAuditedTestEntity;

import static junit.framework.Assert.assertEquals;
import static junit.framework.Assert.assertTrue;

/**
 * A test which checks the correct behavior of AuditReader.isEntityClassAudited(Class entityClass).
 *
 * @author Hernan Chanfreau
 * @author Michal Skowronek (mskowr at o2 dot pl)
 */
public class HasChangedAPITest extends AbstractModifiedFlagsEntityTest {
    public void configure(Ejb3Configuration cfg) {
        cfg.addAnnotatedClass(AuditedTestEntity.class);
        cfg.addAnnotatedClass(NotAuditedTestEntity.class);
    }

    @Test
    @Priority(10)
    public void initData() {
        EntityManager em = getEntityManager();
        em.getTransaction().begin();
        AuditedTestEntity ent1 = new AuditedTestEntity(1, "str1");
        NotAuditedTestEntity ent2 = new NotAuditedTestEntity(1, "str1");

        em.persist(ent1);
        em.persist(ent2);
        em.getTransaction().commit();

        em.getTransaction().begin();

        ent1 = em.find(AuditedTestEntity.class, 1);
        ent2 = em.find(NotAuditedTestEntity.class, 1);
        ent1.setStr1("str2");
        ent2.setStr1("str2");
        em.getTransaction().commit();
    }

    @Test
	public void testHasChangedHasNotChangedCriteria() throws Exception {
		List list = getAuditReader().createQuery().forRevisionsOfEntity(AuditedTestEntity.class, true, true)
                .add(AuditEntity.property("str1").hasChanged()).getResultList();
		assertEquals(2, list.size());
		assertEquals("str1", ((AuditedTestEntity) list.get(0)).getStr1());
		assertEquals("str2", ((AuditedTestEntity) list.get(1)).getStr1());

		list = getAuditReader().createQuery().forRevisionsOfEntity(AuditedTestEntity.class, true, true)
                .add(AuditEntity.property("str1").hasNotChanged()).getResultList();
		assertTrue(list.isEmpty());
	}

}

redpajama_set_name: RedPajamaGithub


Los Angeles and WWE SummerSlam!
Last Friday immediately after work I hopped on the BART straight to SFO to catch a flight to LA for the weekend.
Why LA? Because the beau was there for work, flights were cheap ($160 round trip) and I could just stay in his hotel room! So all I really had to pay for was my flight and my food.
Where I spent most of my Saturday and Sunday morning and afternoon.
I got in to LA late on Friday (thanks United!) and was starving. Luckily, since Rich and I were staying in LA Live, there were still a decent amount of places open and so we headed over to The Yard House not far from our hotel and got some food and I got to try two new beers, Tart Lychee (Lips of Faith) from New Belgium Brewing and Pranqster by North Coast Brewing.
We got up somewhat early on Saturday (10:30, hah) and went to get some breakfast at the Starbucks nearby before Rich had to head off to do the WWE '13 Roster Reveal and I headed to the rooftop pool to work on my tan.
Rich and I met up later to get a quick lunch before he had to run off to a WWE '13 press conference, which he tweeted updates and pics from. While he was there I spent my time taking a long, relaxing, scorching hot shower (best part about hotels is the hot water almost never runs out) and doing my hair and makeup. Then I headed on over to the post-conference WWE '13 event!
The WWE '13 event was awesome. There were a ton of WWE superstars there (almost all of whom I was too shy to go and meet). I saw Mike Tyson as he was leaving the building and Stone Cold Steve Austin walked right by me at one point without me realizing it until Rich said "You know who just walked RIGHT by you, right?" Among the others in attendance were CM Punk, Sheamus, AJ Lee, Brodus Clay and a few others that I can't remember. Oh and Paul Heyman, who is SUPER sweet and super awesome. Unfortunately Kane and Daniel Bryan, my two favorites, were not there.
Paul Heyman, super awesome guy!
Rich played a bit more of the game before I dragged him over to the dessert buffet so that I could try one of everything (little cupcakes so good). I got another drink and relaxed while Rich headed off to do another quick interview with one of the game's developers, Cory, and while he played a match against one of the other developers, Bryan.
Stayed at the event until they were closing the venue down (which really wasn't all that late, around 10:30). Met a lot of cool people, a lot of who were impressed that I like wrestling! I was actually a HUGE WCW fan back in the 90s, but when WCW stopped existing I kind of lost interest. Rich has been the one to get me back into it with his enthusiasm. I have a newfound love for professional wrestling, what with the amount of planning that goes into each match for both storyline and marketing aspects. It's an art, for sure.
Oh, and Rich got a swag bag with WWE '13 hoodies for both of us and a Sheamus Brawlin' Buddy!
Sheamus and I! He makes noises when you throw him!
Sunday I wasn't expecting to do much except spend more time by the pool and have lunch with Rich and his parents. As far as I knew at that point Rich wasn't able to get a +1 for me to go to SummerSlam, so I figured I'd be spending my evening by the pool and watching TV in the room before flying out late that night.
But, Rich had been given a +1 just that day! I was so excited! After our delicious sushi dinner with his parents I hopped right into the shower so that I could get my hair and makeup done in time and then we headed on over to the Staples Center!
Found this abandoned sign on our way to the Staples Center. Was too perfect to not get a pic!
Daniel Bryan entering before the Kane/Bryan match!
Kane entering! You don't realize how loud the explosions are until you're there in person! So loud!
A picture of The Miz's AWESOME blow up sign!
CM Punk! Best in the world!
Kevin Rudolf performing "Don't Give Up"
Triple H entering before his match with Brock Lesnar!
I won't bother to spoil any of the matches for those of you who haven't caught up on your WWE news since Sunday. I loved the Kane vs Bryan match, even though it was hard for me to choose who I wanted to win. The Triple Threat match with CM Punk, Cena and Big Show was also great. Tried to buy a Daniel Bryan "YES!" shirt afterwards, but they were out of everything smaller than a Large, which would be way too big for me, so I ended up just buying one online instead.
Headed back to the hotel to pack up my things quickly, say my goodbyes and head off to the airport and back to San Francisco.
While it might have been a short vacation it was just the recharge I needed.