Journal: Metabolites

Article Title: What Has Longitudinal ‘Omics’ Studies Taught Us about Irritable Bowel Syndrome? A Systematic Review

doi: 10.3390/metabo13040484

Figure Lengend Snippet: The characteristics and key findings of the studies reviewed (arranged alphabetically by the first author’s last name).

Article Snippet: Ankersen, 2021 [ ] , Denmark , 34 IBS patients (either IBS-D or IBS-M) randomised to receive either web-based low FODMAP diet intervention or probiotic; Rome III criteria for diagnosis , Stool sample , CeGaT GmbH culture-independent, whole-genome, shotgun metagenomic, next-generation sequencing of genomic DNA , Four time points, during the course of 1 year , - web-based low FODMAP diet intervention and probiotic intervention were both efficacious in managing IBS symptoms - in both cases, treatment response could not be predicted or explained by the composition of host gut microbiota.

Techniques: Biomarker Discovery, Microarray, Real-time Polymerase Chain Reaction, Functional Assay, Expressing, Next-Generation Sequencing, Bacteria, Gene Expression, Genome Wide, Probiotics, Isolation, Amplification, Sequencing, Starch, Fluorescence, FACS, Concentration Assay, Control, Clinical Proteomics, RNA Sequencing, Modification

Journal: Frontiers in Immunology

Article Title: A Systematic Review of Tissue and Single Cell Transcriptome/Proteome Studies of the Brain in Multiple Sclerosis

doi: 10.3389/fimmu.2022.761225

Figure Lengend Snippet: An overview of the advantages and disadvantages of the omics techniques.

Article Snippet: Epigenomics , Molecular changes on the DNA , Assessment of variability of factors that regulate the genome without changing the DNA sequence , WGBS (whole genome bisulfite-treated DNA sequencing) , Methylome-wide pattern and alterations , Moderate , Complex data analysis, lack of functional knowledge on methylation at other sites , Whole methylome state on single base pair level.

Techniques: Sequencing, Genome Wide, DNA Sequencing, Functional Assay, Methylation, Microarray, Hybridization, Immunoprecipitation, DNA Binding Assay, Binding Assay, Gene Expression, Amplification, Expressing, Clone Assay, Multiplexing, Protein-Protein interactions, Blocking Assay, Microscopy, Activity Assay, Mass Spectrometry, Mass Cytometry

Journal: bioRxiv

Article Title: High levels of DNA replication initiation factors indicate ATRi sensitivity via excessive origin firing

doi: 10.1101/2025.02.27.640046

Figure Lengend Snippet: ( A ) Quantification of the average number of γH2AX foci in replicating cells (EdU+) and ( B ) of the micronuclei rate in RPE hTERT cells treated with DMSO, APH 0.4uM, ATRi 0.5 µM or both for 24h. Results were obtained from three independent immunofluorescence replicates. Paired t-test analysis. ( C ) Western Blot of Cyclin E and β-tubulin proteins in FNE1 cells overexpressing Cyclin E, induced with 2 µg/mL of doxycycline for 0h, 24h or 48h, and Ovsaho ovarian cancer cell line. ( D ) Representation of the protocol used to perform the sensitivity assay to ATR inhibitor using the incucyte microscopy. Cells were treated 24h after seeding and the cell confluence was measured every few hours for 72h. ( E ) Quantification of the confluence over time in response to different doses of ATRi in eight different breast cancer cell lines. Multiple paired t-test analysis. ( F ) Quantification of the mRNA expression (normalized transcripts per million) of CCNE1, CCNE2 and Myc in sensitive and resistant breast cancer cell lines using publicly available data from CCLE. Sensitivity was determined using the GDSC2 dataset, the top 25% of cell lines were considered resistant and the bottom 25% as sensitive. Unpaired t-test analysis. ( G ) Cell confluence dose response of ceralasertib (ATRi) was measured at 72h post-treatment in three KTB immortalized cell lines. Fold confluence of ATRi treated samples was measured related to DMSO at 72h endpoint. Results are representative of at least two independent replicates. ( H ) Quantification of the confluence over time in response to different doses of ATRi in three different KTB breast cell lines. ( I ) Schematic representation of the DNA copy number clustering strategy based on Whole Exome Sequencing data of a large panel of breast cancer cell lines. Multiple paired t-test analysis. Ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Using DNA CNA analysis from Oxford Nanopore long-read whole genome sequencing, we observed that all lines exhibited chaotic genomes with gains and losses of whole and partial chromosomes ( ).

Techniques: Immunofluorescence, Western Blot, Sensitive Assay, Microscopy, Expressing, Sequencing

Journal: bioRxiv

Article Title: High levels of DNA replication initiation factors indicate ATRi sensitivity via excessive origin firing

doi: 10.1101/2025.02.27.640046

Figure Lengend Snippet: ( A,B ) Cell confluence after 72h at the doses of ceralasertib (ATRi) indicated in RPE1 hTERT co-treated with DMSO or Aphidicolin 0.4µM (A) or FNE1 hTERT cells overexpressing CCNE1 by tetracycline induction 48h pre-ATRi treatment (B). Multiple paired t-test analysis. ( C ) Table indicating the genetic and subtype details of a panel of breast cancer cell lines. ( D ) Cell confluence after 72 h at the doses of ceralasertib (ATRi) indicated in the breast cancer cell lines. For A), B) and D) fold confluence of ATRi treated samples was measured relative to DMSO at 72h. Results are representative of three independent experiments for every cell line. HCC1806, HCC1569 and BT-549 are classified herein as the most sensitive, HCC1187 and BT-474 as intermediate and T47D, MDA-MB-231 and BT-20 most resistant. ( E ) Representation of the genomic copy number plots obtained from long read nanopore sequencing. The x-axis represents the 22 chromosomes and the y-axis the copy number. ( F,G ) Quantification of the fraction of genome altered (F) and the number of copy number segments (G) of the sensitive and resistant cell lines. Unpaired t-test analysis. ( H ) Schematic representation of the replication stress-associated CNA features approach. ( I ) Quantification of the normalized CNA value for three different CNA features: loss, gain, and proximity to large, expressed genes, into three different clusters. ( J ) Quantification of the IC50 in µM across the copy number features-defined clusters, using IC50 values from GDSC2 datasets. Unpaired t-test analysis. Ns, not significant; *, P < 0.05; **, P < 0.01.

Article Snippet: Using DNA CNA analysis from Oxford Nanopore long-read whole genome sequencing, we observed that all lines exhibited chaotic genomes with gains and losses of whole and partial chromosomes ( ).

Techniques: Nanopore Sequencing

Journal: bioRxiv

Article Title: High levels of DNA replication initiation factors indicate ATRi sensitivity via excessive origin firing

doi: 10.1101/2025.02.27.640046

Figure Lengend Snippet: ( A ) Schematic representation of the method to generate replication fork dynamics via nucleotide analogue incorporation, nanopore long read sequencing and DNAscent algorithms. Quantification of the fork ( B ) velocity (kb per minute), ( C ) the median fork velocity (kb per min), ( D ) the fork stalling probability and ( E ) the proportion of impeded forks (with a stall score probability above 0.8) in sensitive and resistant cell lines treated either with DMSO or 1h or 24h of 0.5 µM ATRi. (B,D) Mann & Whitney analysis between each ATRi and DMSO per cell line. (C,E) Unpaired t-test analysis between three sensitive and three resistant cell lines. Note that for this experiment, only the DMSO condition was performed for HCC1806. ( F ) Representative microscopy images of a normal anaphase, an anaphase bridge and a lagging chromosome. Chromatin visualized with DAPI. ( G ) Quantification of the segregation error rate in cells treated with DMSO or ATRi 0.5 µM for 72 hours. ( H ) representative image of an interphase cell with a micronucleus. ( I ) Quantification of the micronuclei rate in cells treated with DMSO or ATRi 0.5 µM for 72 hours. Results represent the average of three sensitive cell lines compared to three resistant cell lines with at least two independent replicates per cell line. Paired t-test analysis. Ns, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Article Snippet: Using DNA CNA analysis from Oxford Nanopore long-read whole genome sequencing, we observed that all lines exhibited chaotic genomes with gains and losses of whole and partial chromosomes ( ).

Techniques: Sequencing, MANN-WHITNEY, Microscopy

Journal: bioRxiv

Article Title: High levels of DNA replication initiation factors indicate ATRi sensitivity via excessive origin firing

doi: 10.1101/2025.02.27.640046

Figure Lengend Snippet: ( A ) Schematic representation of the mass spectrometry method to obtain total proteome and phosphoproteomes of breast cancer cell lines. ( B ) Heatmap plot showing the top hit significantly enriched proteins in sensitive compared to resistant cell lines. The orange box shows CDC45 protein. Significantly changing proteins were identified using a one-way ANOVA, with post-hoc score calculation (FDR < 0.05). ( C ) Volcano plot representation of the kinase activities most enriched in sensitive and resistant breast cancer cell lines, obtained by kinase enrichment analysis of phosphoproteomics data measured by mass spectrometry. ( D ) Schematic representation of the enrichment of kinases CDK2 and CDC7 in sensitive cell lines at baseline conditions, exemplified by an increase of phosphorylation of MCM2 at sites S108, S27 and S139, involved in origin firing. ( E ) Quantification of kinase activities enriched in response to 24 hours of 0.5 µM ATRi in sensitive cell lines compared to resistant, Z-scores higher than 1.5. ( F ) Schematic representation of the enrichment of kinases ATM and ATR via activation of substrates MCM2 S108, γH2AX-S140 and p53 S15 involved suggesting a strong activation of the DNA damage response pathway. ( G,H ) Quantification of the replication origins ( G ) and fork density per megabase ( H ) in breast cancer cell lines treated with DMSO or 0.5 µM ATRi for 1 hour. Data obtained from R9 long-read DNA sequencing. ( I ) Quantification of the replication origins and fork density per megabase in sensitive HCC1806 cells, either with DMSO, 0.5 µM ATRi or 1 µM CDC7i or combination for 1 hour. Data obtained from R10 long-read DNA sequencing. ( J ) Quantification of the mean EdU intensity in replicating cells (EdU+) in HCC1806 (sensitive cell line) cells treated with DMSO, 1 µM CDC7 inhibitor (XL-413), 0.5 µM ATRi or the combination for 1 hour. EdU intensity is used as a metric of activation of origin firing. Results presented are representative of two independent replicates with 250 to 500 EdU positive cells quantified per condition. Paired t-test analysis. ( K ) Quantification of the fork velocity (kb per minute) in sensitive HCC1806 cells treated either with DMSO, 0.5 µM ATRi or 1 µM CDC7i or combination for 1 hour. Data obtained from R10.4.1 long-read DNA sequencing (see methods). Mann & Whitney analysis between each treatment condition and DMSO. ( L,M ) Quantification of the dose response to ATRi in combination with DMSO or 0.25 µM of CDC7i in HCC1806 (L) and HCC1569 (M) sensitive breast cancer cell lines. Multiple paired t-test analysis. ( N ) Microscope imaging of γH2AX pan-nuclear staining in the cell population. ( O ) Quantification of γH2AX intensity in replicating cells (EdU+) in response to DMSO or 0.5 µM for 24 hours in breast cancer cell lines, KTB cells and RPE cells as indicated. Paired t-test analysis. ( P ) Quantification of the percentage of cells with pan-nuclear γH2AX staining in the three sensitive cell lines treated with DMSO or 0.5 µM ATRi for 24 hours. Paired t-test analysis. ( Q ) Quantification of the green Cytotox confluence, live death marker, as a fold ratio to DMSO in HCC1806, HCC159, BT-549 and T47D treated with DMSO or 72h ATRi (0.4 µM, 0.8 µM, 1.6 µM, 3.2 µM). Paired t-test analysis. Ns, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Article Snippet: Using DNA CNA analysis from Oxford Nanopore long-read whole genome sequencing, we observed that all lines exhibited chaotic genomes with gains and losses of whole and partial chromosomes ( ).

Techniques: Mass Spectrometry, Phospho-proteomics, Activation Assay, DNA Sequencing, MANN-WHITNEY, Microscopy, Imaging, Staining, Marker

Journal: bioRxiv

Article Title: High levels of DNA replication initiation factors indicate ATRi sensitivity via excessive origin firing

doi: 10.1101/2025.02.27.640046

Figure Lengend Snippet: ( A ) TMT reporter (A.U.) of DNA replication initiation factors CDC45, ORC1, KAT7 and WRNIP1 in sensitive and resistant breast cancer cell lines. Unpaired t-test analysis. ( B ) Quantification of the mRNA expression of CDC45 gene in sensitive and resistant breast cancer cell line using publicly available data from CCLE. Unpaired t-test analysis. ( C ) Quantification of the enriched kinase activities (z-score) in Resistant compared to Sensitive breast cancer cell lines. In red are the kinase activities enriched in resistant and in blue in sensitive cell lines. ( D ) Quantification of the kinase activities enriched in sensitive and resistant cell lines enriched in 0.5 µM ATRi for 1 hour or 24 hours compared to DMSO. ( E ) Quantification of the replication origins and forks density per megabase, fold increase to DMSO, in breast cancer cell lines treated with DMSO, 0.5 µM ATRi for 1 hour or 24 hours. Data obtained from R9 long-read DNA sequencing. Paired t-test analysis. ( F ) Quantification of the proportion of death cell confluence using a green cytotox live death marker in HCC1806 cells in response to ATRi only or to ATRi and 0.25 µM CDC7i. Results are measured after 72 hours of treatment and the fold change is measured for each ATRi treatment to the DMSO and are representative of two independent experiments. ( G,H ) Quantification of the dose response to ATRi in combination with DMSO or 0.25µM of CDC7i in T47D (F) and MDA- MB-231 (G) resistant breast cancer cell lines. ( I ) Quantification of the number of γH2AX foci per replicating cells (EdU+) in breast cancer, KTB and RPE1 cell lines treated with DMSO or 0.5 µM ATRi for 24 hours. Paired t-test analysis. ( J ) Quantification of the γH2AX intensity per replicating cell (EdU+) in breast cancer cell lines treated with DMSO or 0.5 µM ATRi for 24 hours. Paired t-test analysis. Ns, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Article Snippet: Using DNA CNA analysis from Oxford Nanopore long-read whole genome sequencing, we observed that all lines exhibited chaotic genomes with gains and losses of whole and partial chromosomes ( ).

Techniques: Expressing, DNA Sequencing, Marker