The metabolite annotation and integration were done with the TraceFinder 5.1 software (Thermo Fischer Scientific) using confirmed retention times by in-house standard library (MSMLS-1EA, Merck) and their m/z.
| Storage Condition | Details | Temperature Range |
|---|---|---|
| Cool, Dry Place | Keep away from light and moisture | 15-30°C |
| Keep Out of Reach of Children | To prevent accidental ingestion | - |
| Packaging | Original tightly sealed containers | - |
The data quality was monitored throughout the run using pooled QC sample prepared by pooling 5 μL from each suspended sample and interspersed throughout the run every 10 samples. The data was quality controlled for peak quality (poor chromatography), prefiltered with 20 % RSD cutoff of the pooled QC and noise.
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5.1.0).139 Filtered DGEs were imported using the function “Read10x” and only genes detected in at least 5 cells were kept for downstream analyses. Similarly, cells with less than 200 genes and 3,000 unique transcripts or more than 0.5 % mitochondrial transcripts were discarded. After merging all samples in a single Seurat object, we performed gene expression normalization and scaling for each sample independently using SCTransform.140 We performed unbiased clustering and dimensionality reduction using the first 20 principal components on all samples together. Clusters were manually annotated in 7 cell types analyzing their marker genes (Table S1). To identify differential genes induced by MT-ATP6 variants and sildenafil treatment in a robust and reproducible way, we performed a pseudo-bulk analysis using the DESeq2 package140 (v.
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7.5.1), as described previously.141 Pseudo-bulk analysis was used to determine differentially expressed genes within the individual populations (Table S1). For each population, we generated a pseudo-bulk expression profile for each organoid by summing the expression of 250 randomly selected cells. We sildenafil citrate tablet filtered genes with less than 50 counts in at least 2 samples, normalized expression values by library size, estimated negative binomial dispersions. We performed different comparisons: i) LS + DMSO vs. controls + DMSO (disease signature), ii) LS + sildenafil vs.
RV revealed early hypertrophic molecular changes similar to LV during LV pressure-overload, and sildenafil inhibited these in both ventricles
LS + DMSO (sildenafil signature), iii) controls + sildenafil vs. controls + DMSO (negative control test). For all comparisons, we identified differentially expressed genes and adjusted p value was calculated using Bonferroni correction for multiple testing correction. To estimate glycolytic pathway activity in single cells, we leveraged the AUCell package (v. 1.26.0)142 and the ‘Glycolysis’ gene set in the ‘Hallmark’ database accessed from the MsigDB package (v 7.5.1).143 Plots were generated with ggplot2 (v. We identified metabolites that were differentially present within samples (Table S1). We performed multi-omics integration as previously described.55 Briefly, we integrated bulk transcriptomics, proteomics, and metabolomics datasets derived from LS NPCs and control NPCs (disease signature), as well as transcriptomics and metabolomics datasets from sildenafil-treated LS NPCs and untreated LS NPCs (sildenafil signature). For both cases, we used all significantly deregulated molecules (adjusted p-value ≤ 0.05) as input lists for the OmicsNet platform (v.2.0) and retrieved their interactions based on STRING (confidence score 0.7) and Recon3D databases. Molecular interactions were filtered with the Prize-Collecting Steiner Forest (PCSF) algorithm, retaining only the most informative nodes regarding network organization and functionality.
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For network visualization, we chose the Organic Layout from yFiles plugin in Cytoscape.
- Side effects at high doses like 100g can include vision impairment and hearing loss.
- Sildenafil can cause headaches, dizziness, or temporary vision changes.
- Patients taking medications for blood pressure should consult their doctor beforehand.
- The drug's effect lasts around 4-6 hours, but duration varies.
- Misuse or overdose increases the risk of serious health complications.
To dissect the rescue mechanism of sildenafil, we searched for molecules with reversed expression in the disease signature and sildenafil signature datasets.
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3.5.1) and piping using dplyr (v. 1.1.4) and visualized with uniform manifold approximation and projection (UMAP). We carried out proteomics using mass spectrometry (MS) for four MT-ATP6 mutant NPC lines (ATP6_2, ATP6_4, ATP6_5, and ATP6_7) and four healthy control NPC lines (CTRL_1, CTRL_2, CTRL_3, and CTRL_4) with n=3 biological replicates per condition that were treated with either DMSO or 10 μM sildenafil in DMSO for 16 h. Cells were washed three times with ice-cold DPBS, detached using a cell scraper, transferred to pre-chilled 1.5 ml tubes and centrifuged at 1,000-3,000 rpm for 10 min at 4 °C. Subsequently, the supernatant was removed, and intact cells were snap-frozen.
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Lysates were denatured at 95 °C for 10 min shaking at 1,000 rpm in a thermal shaker and sonicated in a water bath for 10 min. The protein concentration of each sample was measured with a BCA protein assay kit (23252, Thermo Fisher Scientific). 500 ng protein was used per sample and diluted with a dilution buffer containing 10 % acetonitrile and 25 mM Tris-HCl, pH 8.0, to reach a 1 M GdmCl concentration. Then, proteins were digested with LysC (Roche; enzyme to protein ratio 1:50, MS-grade) shaking at 800 rpm at 37 °C for 3.5 hours. The digestion mixture was diluted again with the same dilution buffer to reach 0.5 M GdmCl, followed by tryptic digestion (Roche, enzyme to protein ratio 1:50, MS-grade) and incubation at 37 °C overnight in a thermal shaker at 800 rpm.
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Peptides were acidified with formic acid to a final concentration of 2 %. LC-MS/MS was performed by nanoflow reversed-phase liquid chromatography (Dionex Ultimate 3000, Thermo Fisher Scientific) coupled online to a timsTOF SCP mass spectrometer (Bruker Daltonics) using the data-independent acquisition (DIA) method with parallel accumulation serial fragmentation (PASEF). 150 ng of each desalted digest was applied to the column and peptides were eluted using a gradient of 3.8 to 38 % solvent B in solvent A over 60 min (total run time) at a flow rate of 400 nl per minute. The sildenafil 12 5 mg "match between run" feature was used and the mass search range was set to m/z 400 to 1,000. MS data were processed with Dia-NN (v1.8.1) and searched against an in silico predicted human spectral library (Table S1). Molecules were annotated in the REACTOME and GeneOntology databases for pathway and biological processes enrichment analyses, respectively, using the clusterProfiler package (v4.10.0).
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Transcript counts were determined using Salmon (version 1.10.1). DESeq2 (version 1.40.2)137 was used to identify differentially expressed genes in the NPC samples or in brain organoid samples (Table S1) (Benjamini-Hochberg-adjusted p-value 0.05). Gene Ontology (GO) enrichment analysis was performed to identify biological processes (BP) and cellular components (CC) using the enrichGO function within ClusterProfiler (4.8.3), using the annotations of org.Hs.eg.db (version 3.18.0). Visualizations were generated with ggplot2 (version 3.5.1). For single-nucleus RNA sequencing (snRNAseq), we used day 72 brain organoids generated with the second protocol138 from control iPSCs (CTRL1) and LS iPSCs (ATP6_7).
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Brain organoids were treated with either DMSO or 10 μM sildenafil resuspended in DMSO for 45 days from day 27 to day 72. We used 15 pelleted organoids per sample (n=8 samples) with n=2 biological replicates per condition (n=120 cortical brain organoids in total). snRNA-seq was performed using the 10X Genomics Chromium system, following the manufacturer's protocol. Nuclei were isolated using mechanical dissociation with gradient centrifugation and stained with DAPI to assess nuclear integrity. Barcoding and cDNA amplification were carried out using the Chromium Single Cell 3' (vNext) Reagent Kit.
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Libraries were prepared and sequenced on the Illumina NovaSeq 6000, generating paired-end reads of 100 bp. Data processing, including demultiplexing, alignment to the reference genome (GRCh38-2020-A), and gene quantification, was performed using Cell Ranger (v7.2.0) from 10x Genomics with default parameters. To generate digital gene expression (DGE) matrices for each sample. DGEs were further processed in R (v. 4.4.1) using Seurat (v. To explore the regulatory effect of sildenafil in gene expression levels, we retrieved the drug’s signatures from the L1000 FWD database.
- Sildenafil 100g refers to a high dosage of the medication used to treat erectile dysfunction.
- This dosage is significantly higher than the common 50mg or 25mg tablets.
- Always consult a healthcare provider before using sildenafil in such high amounts.
- Overuse can cause severe side effects like dangerous drops in blood pressure.
- Sildenafil 100g is typically used only under strict medical supervision for specific cases.
Genes that had a consistent pattern of re-regulation across all examined cell lines after sildenafil treatment were considered as literature-indicated drug targets.
- Sildenafil 100g is rarely prescribed and considered an experimental or off-label dose.
- Like all medications, it should only be used as directed by a healthcare professional.
- Overdose signs include severe hypotension, vision loss, or chest pain.
- Use with caution in patients with liver or kidney impairment.
- Avoid alcohol consumption when using sildenafil, especially at high doses.
We evaluated these literature-indicated drug targets in our transcriptomics datasets by filtering those genes that had the same pattern of re-regulation in LS NPCs after sildenafil administration (LS + sildenafil vs. LS + DMSO).
- Sildenafil was first approved for erectile dysfunction treatment in the late 1990s.
- Its effectiveness is dose-dependent, but higher doses do not always mean better results.
- Overdosing may lead to priapism, a painful and prolonged erection requiring medical help.
- The drug is not intended for recreational use and misuse can be dangerous.
- Individuals with heart conditions should avoid high doses like 100g without medical approval.
A final list of literature-indicated drug targets with a similar regulation pattern in treated LS NPCs was considered a cross-validated set of genes involved in the mechanism of action of sildenafil.
Sildenafil Enhances Renal Blood Flow but Does Not Protect Against Ischaemia Reperfusion Injury in a Model of Donation after Circulatory Death Kidney Transplantation
Metabolites were extracted from cells with 500 μl of cold extraction solvent (Acetonitrile:Methanol:MilliQ; 40:40:20, Thermo Fischer Scientific). Subsequently, samples were processed with three cycles of sonication (60 s) and vortexing (120 s) followed by centrifugation at 14,000 rpm at 4 °C for 5 min. Next, the samples were centrifuged, the supernatants transferred to evaporation tube and evaporated to dry under nitrogen stream. Samples were reconstituted in 40 μl extraction buffer (Acetonitrile:Methanol:MilliQ; 40:40:20) and transferred to LC-MS vials. 2 μl of the samples were analyzed with Thermo Vanquish UHPLC coupled with Q-Exactive Orbitrap mass spectrometer equipped with a heated electrospray ionization (H-ESI) source probe (Thermo Fischer Scientific).
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A SeQuant ZIC-pHILIC (2.1 × 100 mm, 5 μm particle) column (Merck) was used for chromatographic separation. The gradient elution was carried out with a flow rate of 0.1 ml/min and mobile phase gradient with 20 mM ammonium hydrogen carbonate, adjusted to pH 9.4 with ammonium solution (25 %) as mobile phase A and acetonitrile as mobile phase B, 0-2 min 80 % B, 2-17 min 80-20 % B, 17-24 min 80 % B. The column oven and auto-sampler temperatures were set to 40 ± 3 °C and 5 ± 3 °C, respectively. Following setting were used for MS: full scan range: 55-825 m/z, polarity switching; resolution of 35,000, the spray voltages: 4250 V for positive and 3250 V for negative mode; the sheath gas: 25 arbitrary units (AU); the auxiliary gas: 15 AU; sweep gas flow 0; capillary temperature: 275 °C; S-lens RF level: 50.0. Instrument control was operated with the Xcalibur software (Thermo Fischer Scientific).