Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A) GDF15 levels; B) ACE2 levels ; C) ratio GDf15/ACE2 ; The box plots represent the maximum and minimum levels (whiskers), the upper and lower quartiles, and the median. The length of each box represents the interquartile range. Dots represent outliers. Statistical significance between groups was determined using the ANOVA test. ** p <0.001, # p <0.001 UCI vs Control, n/s (non significant); GDF15 (Growth differentiation factor 15); ACE2 (angiotensin-converting enzyme 2).

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Control

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: GDF15 is positive associated with age (A), while ACE2 is not correlated with age (B). Each dot represents an individual value. The solid blue line represents the regression line. The grey shade represents the confidence interval.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: The box plots represent the maximum and minimum levels (whiskers), the upper and lower quartiles, and the median. The length of each box represents the interquartile range. Dots represent outliers. Statistical significance between groups was determined using the ANOVA test. ** p <0.001, # p <0.001 UCI vs Control, n/s (non significant); ACE2 (angiotensin-converting enzyme 2).

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Control

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A) SARS-CoV-2 cell entry assay strategy: Lentiviral-based replication-defective pseudovirus were generated in HEK293T cells from lentiviral parental genes, SARS-CoV-2 Spike and encapsulating a mCherry reporter. Since the entry steps of the SARS-CoV-2 pseudovirions are governed by the coronavirus Spike protein at their surface, they enter cells in a similar fashion to native counterparts. A549 airway cells were transfected with exogenous GFP-hACE2 enabling SARS-CoV-2 pseudovirus to effectively infect the cells with mCherry reporter. Double-positive GFP/mCherry cells were quantified by flow cytometry to assess viral infection capacity. B,C) Following the strategy described in A, A549 cells expressing GFP-hACE2 were assayed for cell entry by SARS-CoV-2 pseudovirus expressing either empty vector (Δ Spike) or Spike protein corresponding to origin variant (Wuhan-1) or variants-of-concern Alpha, Beta, Delta or Zeta. A representative flow cytometry experiment is shown. Bars demonstrate mean and Standard Error of Mean while each data point represents a unique experiment; ****P < 0.0001; **P < 0.01; *P < 0.1 by t test.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Generated, Transfection, Flow Cytometry, Infection, Expressing, Plasmid Preparation, Variant Assay

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A549 cells were transfected with either GFP-ACE2 WT, GFP-ACE2 polymorphisms or GFP alone. Then, ACE2 protein expression was analyzed by A) Western Blot with MA5-32307 antibody B) Immunocytochemistry (red) with either MA5-32307 antibody (left panel) or MAB933 antibody (right panel). Nuclei was stained with DAPI (blue). Transfected cells contain GFP (green). CT: secondary antibody control to detect unspecific binding. Images were acquired with Cell Observer-Zeiss. Scale bar: 50 μm

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Transfection, Expressing, Western Blot, Immunocytochemistry, Staining, Control, Binding Assay

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A) Studying the effect of ACE2 SNPs. Non-synonymous ACE2 single nucleotide polymorphism were selected among those fulfilling the triple criteria of high allelic frequency (Allele freq > 1.00e-4; Allele count > 20); involved in ACE2-claw S-protein RBD-binding interface and previously associated to clinical outcome. B, C) Following the strategy described in , A549 cells expressing either GFP-ACE2 either WT or polymorphisms were assayed for cell entry by SARS-CoV-2 pseudovirus expressing either empty vector (Δ Spike) or Spike protein corresponding to origin variant (Wuhan-1) or variants-of-concern Alpha, Beta, Delta or Zeta. A representative flow cytometry experiment is shown. Bars demonstrate mean and Standard Error of Mean while each data point represents a unique experiment; ***P < 0.001, **P < 0.01, *P < 0.1, ns P >0.1 to WT by t test.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Binding Assay, Expressing, Plasmid Preparation, Variant Assay, Flow Cytometry

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A) Heatmap showing the distribution of ACE2 variants in the hospitalization severity groups. Coloured squares indicate de presence of the ACE2 variants. Red: Promoting; Green: Protective; Yellow: No-effect. B) Frequencies of ACE2 SNPs among hospitalization severity groups. Bars represent frequencies of the SNP in each group C) Frequencies of ACE2 SNPS among susceptibility groups.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: GDF15 and ACE2 levels across ACE2 variants.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: GDF15 and ACE2 levels among ICU and non-ICU COVID-19 patients and ACE2 genotypes.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: Genotype 0 corresponding to subjects that did not carry any variant; Genotype 1, subjects that carry at least one promoting variant; Genotype 2, subjects that carry at least one protective variant; Genotype 3, subjects that carry at least one promoting and one protective variant ( ; ). We only found differences in the levels of the ACE2 mRNA ( P <0.05).

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Variant Assay

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: Then, PCR1, PCR2 and PCR3 were performed in order to amplify the regions comprising the studied SNPs. PCR products were sequenced and aligned against reference ACE2 (NM_021804). PCR1: residues from Ser3 to Met249 (743 bp), PCR2: residues Phe308 to Arg621 (944 bp), PCR3: residues from Val670 to Val752 (250 bp) A) Agarose gel electrophoresis with the PCR products of several patients was performed to confirm specificity. B) Representative image of the alignment of the sequenced (forward and reverse) PCR products against reference ACE2 using SnapGene® Software. In particular, the image corresponds to the PCR2 of the non-ICU patient 10 that presents the L351V variant.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Agarose Gel Electrophoresis, Software, Variant Assay

Journal: International Journal of Biological Sciences

Article Title: ACE2 negatively regulates the Warburg effect and suppresses hepatocellular carcinoma progression via reducing ROS-HIF1α activity

doi: 10.7150/ijbs.81498

Figure Lengend Snippet: ACE2 suppresses HIF1α activity in HCC. ( A ) GSEA plot showed a close link between ACE2 expression and hypoxia in HCC samples from the TCGA cohort. ( B ) The effects of ACE2 overexpression on HIF1α transcriptional activity in the presence or absence of A779 (1 μM) were measured in SNU-475 and SK-Hep1 cells. ( C ) The effects of ACE2 knockdown on HIF1α transcriptional activity in the presence or absence of Ang-(1-7) (10 -8 M) were measured in HCC-LM3 and Hep3B cells. ( D ) The effects of ACE2 overexpression on the activity of ACE2-associated signaling pathways (p38 MAPK, ERK1/2, JNK, NF-κB, AKT, SHP2, and HIF1α) in SNU-475 and SK-Hep1 cells was analyzed by Western blotting. ( E ) The levels of p-SHP2, SHP2, and HIF1α in sh-Ctrl and sh-ACE2 HCC-LM3 and Hep3B cells was analyzed by Western blotting. ( F ) The effects of ACE2 overexpression on ROS generation in the presence or absence of A779 (1 μM) were measured in SNU-475 and SK-Hep1 cells. ( G ) The effects of ACE2 knockdown on ROS generation in the presence or absence of Ang-(1-7) (10 -8 M) were measured in HCC-LM3 and Hep3B cells. ( H ) The effects of ACE2 knockdown on HIF1α transcriptional activity in the presence or absence of 1 mM N-acetyl cysteine (NAC) treatment were measured in HCC-LM3 and Hep3B cells. *P < 0.05 and **P < 0.01.

Article Snippet: The commercial HIF1α Transcription Factor Assay Kit (ab133104, Abcam) was used to determine the effect of ACE2/Mas receptor/NAC on HIF1α activity.

Techniques: Activity Assay, Expressing, Over Expression, Knockdown, Protein-Protein interactions, Western Blot

Journal: International Journal of Biological Sciences

Article Title: ACE2 negatively regulates the Warburg effect and suppresses hepatocellular carcinoma progression via reducing ROS-HIF1α activity

doi: 10.7150/ijbs.81498

Figure Lengend Snippet: Clinical relevance of ACE2 in HCC samples . ( A ) Representative IHC staining of ACE2, HIF1α or p-SHP2 protein levels in human HCC specimens (n = 202); scale bar, 50 μm. ( B ) Correlation between ACE2 and p-SHP2 protein was determined by Spearman analysis. ( C ) Correlation between ACE2 and HIF1αprotein was determined by Spearman analysis. ( D ) For PDX model, representative IHC staining of ACE2 in two HCC samples was shown. ( E ) The effect of ACE2 on the tumor growth of PDX xenograft was studied by a subcutaneous xenograft model. ( F ) The schematic diagram for the molecular mechanism of ACE2-mediated suppression of HIF1α activity and tumor growth in HCC. In brief, ACE2 metabolizes Ang II to Ang-(1-7), which activates Mas receptor and leads to the phosphorylation of SHP2. SHP2 activation blocks ROS generation, which further stabilizes HIF1α protein. HIF1α acts as a key transcriptional factor to induce the expression of glucose transporters and glycolytic genes and enhances the Warburg effect. Finally, tumor growth of HCC is suppressed by ACE2 due to compromised glycolytic flux. ***P < 0.001.

Article Snippet: The commercial HIF1α Transcription Factor Assay Kit (ab133104, Abcam) was used to determine the effect of ACE2/Mas receptor/NAC on HIF1α activity.

Techniques: Immunohistochemistry, Activity Assay, Phospho-proteomics, Activation Assay, Expressing