Journal: Nucleic Acids Research

Article Title: The fate of notch-1 transcript is linked to cell cycle dynamics by activity of a natural antisense transcript

doi: 10.1093/nar/gkab800

Figure Lengend Snippet: Cell cycle dynamics regulate transcription of nAS25. ( A ) Expression level of nAS25 upon progression from M to G1 phase. Expression of C-myc and ki-67, as reporters for cell cycle, increases upon progression to G1. Visualisation of the molecular beacon specific to nAS25 (Mo.Bn. AS25 , top micrograph) shows inheritance of nAS25 by daughter cells during cytokinesis (bottom micrographs). * indicates two-tailed P -value < 0.01. ( B ) Scatter plot shows distribution of putative E2F1 binding sites in exons 25 and 26 of notch-1 locus. Subsequent ChIP analysis (right gel) confirmed binding of E2F1 to the highlighted region of exon 25 in cycling and G1 phase cells. ( C ) Expression level of nAS25, exon 5 and exon 25 of notch-1 after blocking the E2F1-binding site (in exon 25 as per text) using dCas9 and after cleavage of DNA upstream to this site. ( D ) Expression of nAS25 after pharmacological inhibition of Cdk2 using Roscovitine (cell cycle reporter: c-Myc). Application of Mo.Bn. AS25 to detect free nAS25 revealed higher expression of the antisense transcript in G1-arrested cells (Roscovitine + ) relative to cycling and G0-arrested (S.S.) cells. ( E ) Expression of nAS25 at G1 subsequent to stabilisation of chromatin topology of G0-synchronised cells using TMP/UVA. ( F ) Expression of nAS25, exon 5, exon 25 and Hey-1 after simultaneous targeting of the inherited and intra-cycle nAS25. ( G ) Left bar plots show the level of nAS25 after cycloheximide-mediated inhibition of protein synthesis and the resultant lengthening of G1. Right bar plots show the level of nAS25 after inhibition of CDK-1 using RO3306 and the resultant arrest of cycling cells at G2. ( H ) The proposed model for allocation of degradable pool of notch-1 transcript at G0. ABPOBEC1-mediated editing of notch-1 transcript at G0 is prevented by competitive binding of nAS25 to the transcript. The edited pool of notch-1 is subsequently degraded at G1 via NMD and after activation of UPF-1. While ≈ 50% of the inherited nAS25 (nAS25 F0 ) is edited and degraded, intra-cycle generation of the antisense transcript (nAS25 F1 ) via E2F1 re-established the pre-G1 titer of nAS25 (nAS25 total ). The right schematic image presents a summary of the model regarding coupled activity of nAS25/notch-1. The inherited nAS25 protects full notch-1 at G0 by hybridizing to the transcript. The intracycle generation of nAS25 stops transcription of full notch-1 and is transmitted to daughter cells as a non-edited entity.

Article Snippet: HiFi Cas9 Nuclease and Alt-R ® S.p. dCas9 protein were purchased from IDTDNA.

Techniques: Expressing, Two Tailed Test, Binding Assay, Blocking Assay, Inhibition, Activation Assay, Activity Assay

Journal: JCI Insight

Article Title: COVID-19 and RA share an SPP1 myeloid pathway that drives PD-L1 + neutrophils and CD14 + monocytes

doi: 10.1172/jci.insight.147413

Figure Lengend Snippet: ( A ) Venn diagram illustrating numbers of unique and shared marker genes of ST TREM2 hi and BALF FABP4 + macrophage clusters as described in . Marker genes were identified prior to integration of data sets ( , ) and were calculated using MAST, setting a minimum percentage of cells in clusters expressing each marker to 40%. Genes considered differentially expressed at P < 0.05 after Bonferroni correction. ( B ) Heatmap illustrating scaled, pseudobulk expression of shared upregulated marker genes from ST and BALF clusters indicated in A . ( C ) Split UMAP plots comparing BALF macrophage clusters in health, and in mild and severe COVID-19, illustrating changes in expression of the TAM receptors AXL and MerTK , with their respective preferred ligands GAS6 and PROS1 . Intensity of purple indicates expression level. ( D ) Heatmap illustrating scaled, pseudobulk expression of TAM receptors and associated ligands by each BALF cluster, across patient groups. TAM receptors and their ligands were significantly differentially expressed in severe COVID-19 versus healthy tissues ( P ≤ 0.005), with Bonferroni correction for multiple comparison, as confirmed by MAST.

Article Snippet: Blood samples were centrifuged (600 g /15 minutes) and plasma aliquots were stored at –80°C until analysis by ELISA for SPP1 (BMS2066; Thermo Fisher Scientific), S100A12 (DY1052; R&D Systems), GAS6 (DY885B; R&D Systems), and PROS1 (NBP2-60585; NOVUS Biological).

Techniques: Marker, Expressing, Comparison

Journal: JCI Insight

Article Title: COVID-19 and RA share an SPP1 myeloid pathway that drives PD-L1 + neutrophils and CD14 + monocytes

doi: 10.1172/jci.insight.147413

Figure Lengend Snippet: ( A ) Patients and healthy donors, shown as the following: n = 121 patients with acute pneumonia ( n = 29 community acquired SARS-CoV-2 – pneumonia, n = 29 mild/moderate COVID-19, n = 63 severe COVID-19), convalescent COVID-19 ( n = 41), and healthy controls ( n = 10). Representative images of lung CT scans. ( B ) Plasma levels of SPP1, S100A12, GAS6, and PROS1 in groups as in A . ( C ) Spearman’s rank correlations between SPP1, S100A12, GAS6, and PROS1 plasma levels in patients with acute COVID-19 pneumonia ( n = 92) with demographic and clinical parameters. Each box displays the r value, and an asterisk indicates statistical significance of P < 0.05. ( D ) Plasma levels of SPP1, S100A12, GAS6, and PROS1 in patients with acute COVID-19 pneumonia ( n = 92) stratified based on lung functions measured by PaO 2 /FiO 2 at the time of hospital admission. Severe respiratory failure was defined by PaO 2 /FiO 2 ≤ 200. ( E ) Percentage of acute COVID-19 pneumonia patients ( n = 92) with PaO 2 /FiO 2 ≤ 200 based on high plasma levels of SPP1 (≥108 ng/mL), S100A12 (≥59 ng/mL), GAS6 (≥24 ng/mL), and PROS1 (≥15 μg/mL). ( F ) COVID-19 patient plasma levels of SPP1, S100A12, GAS6, and PROS1 at the time of hospital admission ( n = 92) stratified based on a patient’s subsequent need to be transferred to ICU. ( G ) Percentage of patients with acute COVID-19 pneumonia ( n = 92) transferred to ICU during the hospitalization based on having high levels of SPP1 (≥108 ng/mL), S100A12 (≥59 ng/mL), GAS6 (≥24 ng/mL), and PROS1 (≥15 μg/mL) at the time of hospital admission. ( B , D , and F ) Data are presented as violin plots with median and interquartile range. Asterisk indicates 1-way ANOVA (Kruskal-Wallis test) with Dunn’s correction for multiple comparisons if more than 2 groups were compared ( B ), or 2-sided Mann-Whitney U was used when 2 groups were compared ( B and D – G ). ( H ) Kaplan-Meier analysis of the rate of transfer of COVID-19 patients to ICU based on their cut-off values for SPP1, S100A12, GAS6, and PROS1 at the time of hospital admission.

Article Snippet: Blood samples were centrifuged (600 g /15 minutes) and plasma aliquots were stored at –80°C until analysis by ELISA for SPP1 (BMS2066; Thermo Fisher Scientific), S100A12 (DY1052; R&D Systems), GAS6 (DY885B; R&D Systems), and PROS1 (NBP2-60585; NOVUS Biological).

Techniques: Clinical Proteomics, MANN-WHITNEY

Journal: JCI Insight

Article Title: COVID-19 and RA share an SPP1 myeloid pathway that drives PD-L1 + neutrophils and CD14 + monocytes

doi: 10.1172/jci.insight.147413

Figure Lengend Snippet: ( A ) Representative images of lung CT scans (transversal and sagittal view) of a COVID-19 patient taken during acute pneumonia and during convalescence (68.60 ± 4.36 days after hospital discharge). ( B ) Plasma levels of SPP1, S100A12, GAS6, and PROS1 in paired plasma samples from COVID-19 patients at the time of acute pneumonia and at the convalescent phase ( n = 26). ( C ) Plasma levels of SPP1, S100A12, GAS6, and PROS1 in convalescent COVID-19 patients ( n = 41) stratified based on the severity of prior acute pneumonia and compared with the levels of healthy donors ( n = 10). ( D ) Plasma levels of IL-6 in acute pneumonias and post–COVID-19. ( E ) SPP1, S100A12, GAS6, and PROS1 in convalescent COVID-19 patients ( n = 41) stratified based on suffering ( n = 36) or not ( n = 5) at least 1 of the symptoms (fatigue, musculoskeletal, or respiratory symptoms). ( B ) Data are presented as before-and-after plot. Wilcoxon test on paired samples was used, and exact P values are provided on the graphs. ( C – E ) Data are presented as violin plots with median and interquartile range. Asterisks indicate 1-way ANOVA with correction for multiple comparisons if more than 2 groups were compared, or 2-sided Mann-Whitney U test was used when 2 groups were compared ( C – E ). Exact P values are provided on the graphs.

Article Snippet: Blood samples were centrifuged (600 g /15 minutes) and plasma aliquots were stored at –80°C until analysis by ELISA for SPP1 (BMS2066; Thermo Fisher Scientific), S100A12 (DY1052; R&D Systems), GAS6 (DY885B; R&D Systems), and PROS1 (NBP2-60585; NOVUS Biological).

Techniques: Clinical Proteomics, MANN-WHITNEY