Jade-1S phosphorylation induced by CK1α contributes to cell cycle progression

<p>The PHD zinc finger protein Jade-1S is a component of the HBO1 histone acetyltransferase complex and binds chromatin in a cell cycle-dependent manner. Jade-1S also acts as an E3 ubiquitin ligase for the canonical Wnt effector protein β-catenin and is influenced by CK1α-mediated phosphorylation. To further elucidate the functional impact of this phosphorylation, we used a stable, low-level expression system to express either wild-type or mutant Jade-1S lacking the N-terminal CK1α phosphorylation motif. Interactome analyses revealed that the Jade-1S mutant unable to be phosphorylated by CK1α has an increased binding affinity to proteins involved in chromatin remodelling, histone deacetylation, transcriptional repression, and ribosome biogenesis. Interestingly, cells expressing the mutant displayed an elongated cell shape and a delay in cell cycle progression. Finally, phosphoproteomic analyses allowed identification of a Jade-1S site phosphorylated in the presence of CK1α but closely resembling a PLK1 phosphorylation motif. Our data suggest that Jade-1S phosphorylation at an N-terminal CK1α motif creates a PLK1 phospho-binding domain. We propose CK1α phosphorylation of Jade 1S to serve as a molecular switch, turning off chromatin remodelling functions of Jade-1S and allowing timely cell cycle progression. As Jade-1S protein expression in the kidney is altered upon renal injury, this could contribute to understanding mechanisms underlying epithelial injury repair.</p

Plk1 colocalizes with NPHP1 at the base of the cilium.

<p><b>A</b> Ciliated hTERT-RPE1 (human retinal pigmented epithelial cells and HK2 human kidney cells were stained with antibody to Plk1 (green), acetylated α-tubulin (orange), and γ-tubulin (red), and treated with DAPI to visualize DNA (blue). The scale bar represents 5 µm. <b>B</b> Ciliated hTERT-RPE1 cells and HK2 cells were stained with antibody to acetylated α-tubulin (orange), γ-tubulin (red), to NPHP1 or Plk1 as indicated (green), and with DAPI to visualize DNA (blue). The third row shows merged signals from staining with antibody to Plk1 (orange), NPHP1 (green) acetylated α-tubulin (orange), γ-tubulin (red) and DAPI was used to visualize DNA (blue). The scale bar represents 5 µm.</p

Plk1 is activated during ciliary disassembly but has limited influence on disassembly dynamics.

<p><b>A</b> Immuno blot analysis of whole cell lysates prepared from hTERT-RPE1 grown under starved, ciliated conditions (0h) or at the times indicated following serum treatment to induce ciliary resorption. Ph-Plk1^T210 represents activated Plk1; <b>B, C</b> Quantification of the expression of (<b>B</b>) Ph-Plk1^T210 or (<b>C)</b> total Plk1 normalized to β-actin and relativized to the expression level without serum induction. Results from three independent experiments were calculated. * P<0.05, ***P≤0.005. Error bars represent SE. <b>D</b> Upper panel: Immunofluorescence of starved ciliated hTERT-RPE1 cells treated with the Plk1-inhibitor BI 2536 or with DMSO for 3 hours, then maintained in serum-free medium, or induced for ciliary disassembly with 10% serum-containing medium. Lower panel: Immunofluorescence of serum-starved, ciliated hTERT-RPE1 cells treated with siRNA to Plk1 or with scrambled (scr) control siRNA after 0, 2 and 24 hours of serum addition. Image shows acetylated α-tubulin (green), γ-tubulin (red) and DNA (blue). The scale bar represents 10 µm. <b>E</b> - <b>H</b> Quantification of three independent repetitions of experiment presented in <b>D</b>. <b>E</b> and <b>G</b> Quantification of the percentage of ciliated cells, with an average of 250 cells counted for each condition. <b>F</b> and <b>H</b> Quantification of cilia length, with an average of 50 cilia measured for each condition. * P<0.05, **P<0.01. ***P<0.001. Error bars represent SE.</p

Plk1 directly phosphorylates NPHP1 in vitro.

<p><b>A</b> Alignment of NPHP1 protein sequences from multiple species indicates a conserved candidate Plk1 motif at position T87. <b>B</b> An <i>in vitro</i> kinase assay performed with active Plk1 and recombinant His-fused NPHP1 protein indicates phosphorylation within the NPHP1 N-terminal 205 amino acids. CB, Coomassie Blue.</p

Plk1 associates with NPHP1.

<p><b>A</b> Western blot of immunoprecipitates (IP) or lysates (Lys) from HEK293T cells co-transfected with plasmids expressing V5-tagged NPHP1 and Flag-tagged Plk1 or negative control protein (Eps1–225 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038838#pone.0038838-Habbig1" target="_blank">[13]</a>). β-actin was assessed as a loading control. <b>B</b> Western blot of immunoprecipitates (IP) or cell lysates (Lys) from HEK293T cells co-transfected with plasmids expressing Myc-tagged Plk1 and Flag-tagged NPHP1 or empty Flag vector. <b>C</b> Western blot of immunoprecipitates (IP) or cell lysates (Lys) from HEK293T cells transfected with plasmid expressing Flag-tagged NPHP1 or the negative control protein (Eps1–225 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038838#pone.0038838-Habbig1" target="_blank">[13]</a>). Endogenous Plk1 was detected using a specific antibody against Plk1. <b>D</b> A panel of Flag-tagged NPHP1 derivatives, including truncations, internal deletions and a T87A mutant, was analyzed by co-immunoprecipitation with Myc-tagged Plk1. <b>E</b> Western analysis of immunoprecipitates (IP) or cell lysates (Lys) from HEK293T cells co-transfected with plasmids expressing Myc-tagged Plk1 and Flag-tagged NPHP1 constructs as indicated, or the Flag-tagged control protein (Eps1–225). * indicates immunoglobulin heavy chain.</p

A rapid and feasible tool for clinical decision making in community-dwelling patients with COVID19 and those admitted to emergency departments: The Braden-LDH-HorowITZ Assessment – BLITZ

There is no univocal standardized strategy to predict outcomes and stratify risk of SARS-CoV-2 infected patients, notably in emergency departments. Our aim is to develop an accurate indicator of adverse outcomes based on a retrospective analysis of a COVID-19 database established at the Emergency Department (ED) of a North-Italian hospital during the first wave of SARS-CoV-2 infection. Laboratory, clinical, psychosocial and functional characteristics including those obtained from the Braden Scale—a standardized scale to quantify the risk of pressure sores which takes into account aspects of sensory perception, activity, mobility and nutrition—from the records of 117 consecutive patients with swab-positive COVID-19 disease admitted to the Emergency Medicine ward between March 1, 2020 and April 15, 2020 were included in the analysis. Adverse outcomes included admission to the Intensive Care Unit (ICU) and in-hospital death. Among the parameters collected, the highest cutoff sensitivity and specificity scores to best predict adverse outcomes were displayed by lactate dehydrogenase (LDH) blood value at admission > 439 U/L, Horowitz Index (P/F Ratio) < 257 and Braden score < 18. The estimation power reached 93.6%. We named the assessment BLITZ (Braden-LDH-HorowITZ). Despite the retrospective and preliminary nature of the data, a multidimensional tool to assess overall functions, not chronological age, produced the highest prediction power for poor outcomes in relation to SARS-CoV-2 infection. Further analyses are now needed to establish meaningful correlations between ventilation therapies and multidimensional frailty as assessed by ad-hoc validated and standardized tools

Addition of low molecular weight heparin <i>in vitro</i> affects PlGF measurements in the Elecsys Assay.

<p>Assessment of serum levels after the administration of enoxaparin or vehicle <i>in vitro</i> shows only marginal differences for sFlt-1 (<b>A+B</b>). In contrast PlGF levels appear falsely high after enoxaparin (<b>C+D</b>). Consequently the sFlt-1/PlGF ratio appears decreased (<b>E+F</b>).</p

Clinical outcome.

<p>Clinical outcome.</p

The rate of increase in serum sFlt-1 shows positive correlation with the initial serum sFlt-1 levels.

<p>Regression analysis of the alteration in serum sFlt-1 levels following enoxaparin (fold change) and initial sFlt-1 serum levels yields positive correlation with the Spearman correlation coefficient r = 0.8273 (95%CI = 0.4348 to 0.9556; p = 0.0014).</p

Heparin does not affect sFlt-1 protein complex size but interferes with sFlt-1 binding to negatively charged surfaces.

<p>Serum samples before and after low molecular weight heparin treatment were subjected to velocity gradient centrifugation. Western blotting of fractions 5–18 was performed using Flt-1 specific antibody (<b>A</b>). Serum samples before and after addition of 10 mg/l enoxaparin were subjected to cation exchange chromatography. In non-treated serum samples 70.14% of sFlt-1 binds to the column, whereas the remaining 29. 86% are found in the flow through. After enoxaparin treatment only 56.88% of sFlt-1 is bound and 43.11% appear in the flow through (p = 0.0386) (<b>B</b>).</p