Fig 1: Expression of human AAT protein following intravenous injection of modified mRNA in wild type mouse. Modified mRNA in LNP formulation was administered as a one-time intravenous (tail-vein) injection at 0.5 mg/kg into C57BL/6 mice (n = 3) and liver tissue was collected 1 hour; 24 hours and 48 hours post injection. (a) Liver tissue was stained for human AAT protein, which revealed a robust intracellular expression in hepatocytes followed by accumulation in the sinusoidal space, scalebar 100 µm. Tissues were collected and stained at 1, 2, and 24 hours post injection and was compared to untreated control animal were no human AAT protein was expressed. (b) DAB staining changes were compared between time points (Kruskal-Wallis test, median with interquartile range). (c) RT-qPCR was used to confirm the delivery of AAT encoding mRNA into liver tissue, where AAT signal passed threshold at 27 cycles and no signal was detected in untreated samples.
Fig 2: Urinary biomarker levels. a) Comparison of urine concentrations of CCL-18 between the cancer and non-cancer groups (left panel), low-grade and high-grade (middle panel) and non-muscle invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). b) Comparison of urine concentrations of A1AT between the same groups. Median levels are depicted by horizontal lines within boxes, standard deviations are depicted by bars. Significance (p < 0.05) was assessed by the Wilcoxon rank sum test.
Fig 3: SILAC Proteomic Analysis during Early Stage Hepatic Differentiation(A) Experimental set up of SILAC proteomic analysis. hPSCs were labeled with light and heavy amino acids and differentiated to IH cells in CCC (light-labeled cells) and µF (heavy-labeled cells) to analyze heavy/light proteins ratios (H/L ratio) in conditioned media and lysates.(B) Venn diagram of detected proteins in conditioned media of HE and IH cells derived from H0-193 hiPSCs and collected according to the experimental set up in (A).(C) Histograms of secreted proteins detected in HE and IH samples, according to their H/L ratio. Proteins significantly accumulated in µF and in CCC are indicated in red and blue, respectively. The remaining proteins are shown in gray. Insets: pie plots of the percentage of proteins in these three categories.(D) Functional enrichment analysis within GO-BP categories of proteins significantly accumulated in µF in HE and IH samples reveals extracellular matrix-related categories on top.(E) iBAQ values versus SILAC ratios of proteins significantly accumulated in µF in HE and IH samples. ECM-related proteins are highlighted in dark orange and dark green in HE and IH samples, respectively.(F) Bar plot of iBAQ values of ECM-related proteins significantly accumulated in µF. iBAQ is expressed as percentage of the total amount of ECM-related proteins in HE and IH samples, respectively.(G) Network of the protein-protein interactions derived by previous experimental studies using the techniques indicated in the legend. All ECM-related proteins upregulated in µF were included in the analysis, but only proteins with at least one known interaction are reported.(H) Histogram of proteins identified in IH cells lysates, according to their SILAC ratio. Proteins significantly up- and downregulated in µF are indicated in red and blue, respectively. The remaining proteins are shown in gray. Insets: pie plots of the percentage of proteins in these three categories.(I) Volcano plot of the same proteins shown in (H). Hepatic markers TTR, RBP4, APOB, and AAT, but not AFP, are overexpressed in µF.(J) Functional enrichment analysis of Reactome pathways of proteins up- and downregulated in µF highlights metabolic pathways and DNA transcription as top-ranking categories, respectively.
Fig 4: The investigated serpins reduce SARS-CoV-2 infection by inhibition of TMPRSS2-mediated spike protein cleavage. (A) HEK-293T cells transfected with the indicated expression plasmids for 24 h were infected with SARS-CoV-2 (MOI = 0.1) for 6 h, and viral RNA was measured by qPCR. (B and C) Posttransfection (24 h) HEK293T cells were infected for 2 h (MOI = 1) and then trypsinized, washed with PBS, and lysed, and the RNA was extracted. Levels of viral RNA were quantified from cDNA synthesized with (B) random hexamers (C) or only the forward primer selectively quantifying the negative sense RNA. Data are cumulative of three independent experiments performed in triplicate; mean and SEM are shown, and statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (D) Surface plasmon resonance analysis of TMPRSS2 binding to individual serpins. A 2-fold dilution series of TMPRSS2 ranging from 125 nM down to 7.8 nM over immobilized SERPINE1 with results shown as response units (RU). Binding kinetics for all serpins are summarized to the right, including the natural target for SERPINE1, tissue plasminogen activator (tPA), as a positive control. (E) TMPRSS2-mediated S-protein cleavage in the presence or absence of individual serpins and the known protease inhibitor nafamostat mesylate. Data from three independent experiments were quantified, and a representative blot is shown. (F) The intensity of bands in panel E corresponding to cleaved S-protein was quantified using ImageJ (Fuji) and normalized to S-protein and TMPRSS2 control. Mean values and SEM are shown; statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01). (G) HBEC ALI cultures were preincubated apically with recombinant SERPINE1, SERPINA1, or SERPINC1 and infected with SARS-CoV-2 at an MOI of 0.05. The accumulated viral release from the apical side was quantified by qPCR at the indicated time points (n = 3). Mean and SEM are shown; statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01). (H) The concentrations of apically released SERPINA1 and SERPINE2 from HBEC ALI cultures from both group high and group low were determined by ELISA. The apical secretions were collected at three time points (n = 3). Mean and SEM are shown; statistical significance was calculated by unpaired t test (***, P < 0.001).
Fig 5: Analysis of CCL18 and A1AT biomarker performance in an experimental model. Using the experimental model depicted in Figure 1, urinary levels of CCL18 and A1AT were analyzed by ELISA. The addition of whole blood resulted in an increase in CCL18 as well as an increase in A1AT. Error bars indicate standard deviations. *, significance (p < 0.05) compared to pooled urines from healthy subjects. ^, significance (p < 0.05) compared to corresponding lower concentration.
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