Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Meiosis-Specific Regulation of the Saccharomyces cerevisiae S-Phase Cyclin CLB5 Is Dependent on MluI Cell Cycle Box (MCB) Elements in Its Promoter but Is Independent of MCB-Binding Factor Activity

In proliferating S. cerevisiae, genes whose products function in DNA replication are regulated by the MBF transcription factor composed of Mbp1 and Swi6 that binds to consensus MCB sequences in target promoters. We find that during meiotic development a subset of DNA replication genes exemplified by TMP1 and RNR1 are regulated by Mbp1. Deletion of Mbp1 deregulated TMP1 and RNR1 but did not interfere with premeiotic S-phase, meiotic recombination, or spore formation. Surprisingly, deletion of MBP1 had no effect on the expression of CLB5, which is purportedly controlled by MBF. Extensive analysis of the CLB5 promoter revealed that the gene is largely regulated by elements within a 100-bp fragment containing a cluster of MCB sequences. Surprisingly, induction of the CLB5 promoter requires MCB sequences, but not Mbp1, implying that another MCB-binding factor may exist in cells undergoing meiosis. In addition, full activation of CLB5 during meiosis requires Clb5 activity, suggesting that CLB5 may be regulated by a positive feedback mechanism. We further demonstrate that during meiosis MCBs function as effective transcriptional activators independent of MBP1

An Upstream YY1 Binding Site on the HIV-1 LTR Contributes to Latent Infection

<div><p>During HIV-1 infection a population of latently infected cells is established. This population is the major obstacle preventing total eradication of the virus from AIDS patients. HIV-1 latency is thought to arise by various mechanisms including repressive chromatin modifications. Transcription factors such as YY1 have been shown to facilitate repressive chromatin modifications by the recruitment of histone deacetylases. In this study, we identified a novel binding site for YY1 on the HIV-1 LTR, 120 nucleotides upstream of the transcription start site. We show that YY1 can bind to this site <i>in vitro</i> and <i>in vivo</i> and that binding to the LTR is dissociated upon T cell activation. Overexpression of YY1 causes an increase in the proportion of cells that produce latent infections. These observations, in combination with previous results, demonstrate that YY1 plays a prominent role in controlling the establishment and maintenance of latent HIV-1 provirus in unstimulated cells.</p> </div

YY1 overexpression increases the proportion of virus that maintains latent infection.

<p>Panel <b>A</b>: Schematic representation of expression constructs: wild type YY1 (YY1), a YY1 deletion mutant lacking the HDAC1 interaction region (g/a/k) and vector control (pEFlag) used to produce stable cell lines. Indicated are a histidine rich region (His), the glycine/alanine (GA) and glycine/lysine (GK) rich regions, and the zinc finger domains (ZF) DNA binding domain. Panel <b>B</b>: Immunoblot of cell extracts from stable lines transfected with the YY1 (lane 1), g/a/k YY1 mutant (lane 2) and vector control (lane 3) with α-Flag antibody. Panel <b>C</b>: ChIP was performed on pools of stably transfected Jurkat-tat cells overexpressing YY1, the g/a/k YY1 mutant or the vector control, infected with the wild type reporter virus using α-Flag antibody, and the primer sets as described in Figure 2B. Panel <b>D</b>: Stably transfected Jurkat-tat cells overexpressing YY1, the g/a/k YY1 mutant or the vector control were infected with the wild type pTY-LAI-dsRed reporter virus construct. Flow analysis was performed every 24 hours for 4 days and every week for a month post infection. The % active infection was calculated by dividing the number of cells with active HIV-1 LTR (eGFP and dsRed expressing cells) by the number of infected cells (total eGFP expressing cells).</p

YY1 binds to the HIV-1 LTR near RBEIII.

<p>Panel <b>A</b>: EMSA was performed with Jurkat nuclear extracts and radiolabeled RBEIII probe (core RBEIII site highlighted in red). Antibodies to TFII-I (lane 2), USF1 (lane 3), USF2 (lane 4), or YY1 (lane 5) were added to analyze the complex components. Free probe (FP) is shown in lane 6. Panel <b>B</b>: An extract from SF9 cells infected with a baculovirus expressing YY1 was separated on 12% SDS-PAGE and analyzed by immunoblotting with YY1 antibody (lane 2). Lane 1 contains a high molecular weight protein ladder. Panel <b>C</b>: EMSA was performed with recombinant YY1 produced by baculovirus (lanes 1 and 2) or Jurkat nuclear extracts (lane 4) with the radiolabeled RBEIII probe. The RBF2 complex in lane 4 is shown. YY1 antibody was added to the reaction in lane 2. Free probe is shown in lane 3. Panel <b>D</b>: (Top) EMSA was performed with recombinant YY1 and labeled RBEIII probe (lane 1), unlabeled competitor oligonucleotides were added to the reactions at 100-fold molar excess (lanes 2-10) (Bottom). Sequences of unlabeled competitor probes; 2, a known YY1 binding sequence oligonucleotide from the parvovirus promoter; 3, wild type RBEIII probe; 4-5, three nucleotide substitutions; 6-12, point substitutions of the RBEIII probe; 13 free probe (FP). Non-specific (NS) bands are also shown. Substitutions are indicated in lower case.</p

TFII-I binds to RBEIII constitutively.

<p>Panel <b>A</b>: A representative pool of Jurkat-tat cells infected with the wild type (solid bars) and RBEIII/YY1 mutant (open bars) reporter virus were used for ChIP analysis with TFII-I antibody. Panel <b>B</b>: A representative clone of Jurkat-tat cells infected with the wild type reporter virus was induced with PMA or left untreated (control) and used for ChIP analysis with α-TFII-I antibodies, and the primers specific for RBEIII, the enhancer region (ER) and the RBEI site. * Represents P values ≤ 0.05, ** represents P values ≤ 0.01.</p

YY1 is not bound to actively transcribed HIV-1 LTR in unstimulated Jurkat-tat cells.

<p>Panel <b>A</b>: A representative population of Jurkat-tat cells infected with the wild type reporter virus was sorted using by FACS and populations of cells expressing only eGFP (green) or both eGFP and dsRed (red) were collected and expanded in culture. Panel <b>B</b>: ChIP analysis was performed on the expanded populations of Jurkat-tat cells expressing eGFP (green) or both eGFP and dsRed (red) using YY1 antibodies and the primer sets as described in Figure 2B. * Represents P values ≤ 0.05, ** represents P values ≤ 0.01, *** represents P values ≤ 0.001.</p