Higher COVID-19 pneumonia risk associated with anti-IFN-α than with anti-IFN-ω auto-Abs in children

We found that 19 (10.4%) of 183 unvaccinated children hospitalized for COVID-19 pneumonia had autoantibodies (auto-Abs) neutralizing type I IFNs (IFN-alpha 2 in 10 patients: IFN-alpha 2 only in three, IFN-alpha 2 plus IFN-omega in five, and IFN-alpha 2, IFN-omega plus IFN-beta in two; IFN-omega only in nine patients). Seven children (3.8%) had Abs neutralizing at least 10 ng/ml of one IFN, whereas the other 12 (6.6%) had Abs neutralizing only 100 pg/ml. The auto-Abs neutralized both unglycosylated and glycosylated IFNs. We also detected auto-Abs neutralizing 100 pg/ml IFN-alpha 2 in 4 of 2,267 uninfected children (0.2%) and auto-Abs neutralizing IFN-omega in 45 children (2%). The odds ratios (ORs) for life-threatening COVID-19 pneumonia were, therefore, higher for auto-Abs neutralizing IFN-alpha 2 only (OR [95% CI] = 67.6 [5.7-9,196.6]) than for auto-Abs neutralizing IFN-. only (OR [95% CI] = 2.6 [1.2-5.3]). ORs were also higher for auto-Abs neutralizing high concentrations (OR [95% CI] = 12.9 [4.6-35.9]) than for those neutralizing low concentrations (OR [95% CI] = 5.5 [3.1-9.6]) of IFN-omega and/or IFN-alpha 2

Max1 links MBF dependent transcription upon completion of DNA synthesis in fission yeast

When DNA replication is challenged, cells activate a DNA synthesis checkpoint blocking cell cycle progression until they are able to overcome the replication defects. In fission yeast, Cds1 is the effector kinase of this checkpoint, inhibiting M phase entry, stabilizing stalled replication forks and triggering transcriptional activation of S-phase genes; the molecular basis of this last effect remains largely unknown. The MBF complex controls the transcription of S-phase genes. We have purified novel interactors of the MBF complex and among them we have identified the repressor Max1. When the DNA synthesis checkpoint is activated, Max1 is phosphorylated by Cds1 resulting in the abrogation of its binding to MBF. As a consequence, MBF-dependent transcription is maintained active until cells are able to overcome this challenge.Cuando la replicación del DNA se ve alterada, las células activan un mecanismo de control bloqueando la progresión del ciclo celular hasta que son capaces de superar el daño. En la levadura de fisión, Cds1 es la proteína kinasa efectora de dicha respuesta, mediante inhibición de la entrada en fase M, estabilización las horquillas de replicación bloqueadas, e inducción de la activación de la transcripción de los genes de fase S; siendo la base molecular de este último proceso poco conocida. El factor de transcripción MBF controla la transcripción de los genes de fase S. Hemos purificado proteínas que interaccionan con MBF, y entre ellas, hemos identificado al represor Max1. Cuando el checkpoint de síntesis de DNA es activado, Max1 es fosforilado por la kinasa Cds1, y esto se traduce en la disociación de Max1 del complejo MBF. Como consecuencia, la transcripción MBF-dependiente se mantiene activa hasta que las células son capaces de superar el daño

Roles of CDK and DDK in Genome Duplication and Maintenance: Meiotic Singularities

Cells reproduce using two types of divisions: mitosis, which generates two daughter cells each with the same genomic content as the mother cell, and meiosis, which reduces the number of chromosomes of the parent cell by half and gives rise to four gametes. The mechanisms that promote the proper progression of the mitotic and meiotic cycles are highly conserved and controlled. They require the activities of two types of serine-threonine kinases, the cyclin-dependent kinases (CDKs) and the Dbf4-dependent kinase (DDK). CDK and DDK are essential for genome duplication and maintenance in both mitotic and meiotic divisions. In this review, we aim to highlight how these kinases cooperate to orchestrate diverse processes during cellular reproduction, focusing on meiosis-specific adaptions of their regulation and functions in DNA metabolism

CDK activity provides temporal and quantitative cues for organizing genome duplication.

In eukaryotes, the spatial and temporal organization of genome duplication gives rise to distinctive profiles of replication origin usage along the chromosomes. While it has become increasingly clear that these programs are important for cellular physiology, the mechanisms by which they are determined and modulated remain elusive. Replication initiation requires the function of cyclin-dependent kinases (CDKs), which associate with various cyclin partners to drive cell proliferation. Surprisingly, although we possess detailed knowledge of the CDK regulators and targets that are crucial for origin activation, little is known about whether CDKs play a critical role in establishing the genome-wide pattern of origin selection. We have addressed this question in the fission yeast, taking advantage of a simplified cell cycle network in which cell proliferation is driven by a single cyclin-CDK module. This system allows us to precisely control CDK activity in vivo using chemical genetics. First, in contrast to previous reports, our results clearly show that distinct cyclin-CDK pairs are not essential for regulating specific subsets of origins and for establishing a normal replication program. Importantly, we then demonstrate that the timing at which CDK activity reaches the S phase threshold is critical for the organization of replication in distinct efficiency domains, while the level of CDK activity at the onset of S phase is a dose-dependent modulator of overall origin efficiencies. Our study therefore implicates these different aspects of CDK regulation as versatile mechanisms for shaping the architecture of DNA replication across the genome

Program of DNA replication in cells operating with a single cyclin B-CDK fusion protein.

<p><b>A)</b> Experimental design for determining the replication profiles in Cdc13-Cdc2 and <i>Control</i> cells. Top: Schematic for Cdc13-Cdc2 cells. For array experiments, 12 mM HU was added 10 min after inhibitor removal (Release). Cells were then harvested 60 min following the addition of HU (70 min after release from G2), after S phase is completed in a sample without HU (see <i>B</i>). Bottom: Schematic for <i>cdc25-22</i> cells. For array experiments, HU was added at the time of the return to permissive temperature. Cells were then harvested 90 min following the shift to 25°C, when S phase is completed in a sample without HU (see <i>B</i>). B: Block. <b>B)</b> DNA content analysis of Cdc13-Cdc2 and <i>Control</i> cells undergoing a synchronous S phase. Experimental procedures were as in <i>A</i>, but without HU treatment. These assays allowed the determination of the timing of sampling when using HU. Samples in which cells are undergoing DNA replication are shown in dark gray. Cdc13-Cdc2 cells arrested in G2 with a 2C DNA content (B: Block as in <i>A</i>) enter S phase ~30 min after release from inhibitor treatment. A 4C peak then appears, and cell division occurs while S phase is finishing, resulting in a 2C peak. <i>Control</i> cells initially arrested in G2 with a 2C DNA content (B: Block as in <i>A</i>) enter S phase at ~40 min following the release from G2. This leads to the appearance of a 4C peak, which is resolved upon cytokinesis shortly after the genome is duplicated (profiles prior to S phase and after cell division, which occurs ~100 min after release, are not shown). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007214#sec009" target="_blank">Materials and Methods</a> for additional details for the interpretation of flow cytometry profiles. <b>C</b>) Origin usage profiles of <i>Control</i> (black) and Cdc13-Cdc2 (red) cells. x-axis: chromosome coordinates, y-axis: origin efficiencies. For full detailed profiles, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007214#pgen.1007214.s001" target="_blank">S1A Fig</a>. <b>D)</b> Detailed view of origin efficiencies in a representative region of the genome for <i>Control</i> (black) and Cdc13-Cdc2 (red) cells. Data are as in <i>C</i>. x-axis: chromosome coordinates, y-axis: origin efficiencies. <b>E)</b> Comparison of origin efficiencies in Cdc13-Cdc2 vs. <i>Control</i> cells. The efficiencies of the 598 origins common to both replication programs are shown. x-axis: efficiency in the <i>Control</i> program, y-axis: efficiency in the Cdc13-Cdc2 program. The dashed line represents efficiencies if they were identical in the two backgrounds. <b>F)</b> Regional profiles of replication domains in <i>Control</i> (black, scale on left y-axis) and Cdc13-Cdc2 (red, scale on right y-axis) cells. The averages of origin efficiencies were determined for continuous windows of ~250 kb (1000 probes) across the genome. x-axis: chromosome coordinates, y-axis: average origin efficiencies. Note that for comparison, the Cdc13-Cdc2 profile is shown on a scale that is shifted to adjust for the 4.2% difference in overall average origin efficiency between the two backgrounds (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007214#pgen.1007214.s001" target="_blank">S1B Fig</a>). This does not alter the absolute efficiency scales on the y-axes, which each span 50% efficiency. Replication efficiency domains are clearly apparent: for instance, the right arm of chromosome II (2.5–3.5 Mb) comprises a low efficiency region, whereas the right arm of chromosome I (3–4 Mb) represents a high efficiency region. The Spearman’s rank correlation coefficient (ρ) comparing the replication programs in the two backgrounds is shown and demonstrates a very strong correlation. ***: p-value < 0.001.</p

Limits of reprogramming DNA replication by temporal regulation of CDK activity.

<p><b>A)</b> Experimental design for determining the replication profile in Cdc13-Cdc2 cells with a long delay in CDK availability (G1+165). For array experiments, HU was added to the cells 10 min before the release from the 20 μM 3-MBPP1 inhibitor treatment (B_G1). Cells were harvested 30 min following this release, after S phase is completed in a sample without HU (see <i>B</i>). B_G1: G1 block. <b>B)</b> DNA content analysis of G1+15 and G1+165 cells undergoing a synchronous S phase. The data for G1+15 are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007214#pgen.1007214.g002" target="_blank">Fig 2B</a>. The experimental procedure for G1+165 was as in <i>A</i>, but without HU treatment. Samples in which cells are undergoing DNA replication are shown in dark gray. B_G1 indicates the end of the prolonged G1. In the G1+165 conditions, cells have fully divided by the time of the release (B_G1), resulting in one major 1C peak. The ensuing S phase leads to a rightward shift of this peak to 2C. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007214#sec009" target="_blank">Materials and Methods</a> for additional details for the interpretation of flow cytometry profiles. <b>C)</b> Origin usage profiles of G2B, G1+15, and G1+165. Data for G2B and G1+15 are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007214#pgen.1007214.g002" target="_blank">Fig 2C</a>. x-axis: chromosome coordinates, y-axis: origin efficiencies. For full detailed profiles and one-to-one comparisons of origin efficiencies, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007214#pgen.1007214.s004" target="_blank">S4A and S4B Fig</a>, respectively. <b>D)</b> Regional profiles of replication domains in G2B (black), G1+15 (blue), and G1+165 (green). The averages of origin efficiencies were determined for continuous windows of ~250 kb (1000 probes). Data for G2B and G1+15 are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007214#pgen.1007214.g002" target="_blank">Fig 2F</a>. Dashed lines indicate the average efficiencies of all the origins in each program (G2B: 24.9%; G1+15: 29.4%; G1+165: 33.4%). x-axis: chromosome coordinates, y-axis: average origin efficiencies. <b>E)</b> Regional analysis of the changes in origin efficiencies. Top panel: Differences of the average origin efficiencies (blue: G1+165 - G2B; black: G1+15 - G2B) determined in continuous windows of 1000 probes (~250 kb). Bottom panel: Differences of the average origin efficiencies (green: G1+165 - G1+15) determined in continuous windows of 1000 probes (~250 kb). The dashed line marks the average difference in origin efficiencies between the G1+165 and G1+15 conditions (4%). x-axis: chromosome coordinates, y-axis: difference in average origin efficiencies. The higher efficiencies in G1+165 are likely to be due to the higher CDK activity level in this condition (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007214#pgen.1007214.g005" target="_blank">Fig 5</a>).</p

Strains used in this study.

<p>Strains used in this study.</p

Diagram illustrating the regulation of the replication program through modulation of CDK activity.

<p>Dashed lines: average efficiencies of all of the origins in each program; red lines: regional efficiency profiles of origin usage. Top panel: Changes in the timing at which CDK activity reaches the S phase threshold result in reprogramming of replication efficiency domains. Our results show that a major equalization of origin efficiencies occurs as a consequence of short G1 extensions, while a prolonged delay leads to a minor further alteration of the replication pattern. Bottom panel: Alterations in the level of CDK activity at the G1/S transition generate quantitative changes in origin efficiencies across the entire genome without affecting the domains of efficiencies. In this case, average origin efficiencies vary depending on activity levels.</p