multiplex gene panels  (Natera Inc)

Journal: Science advances

Article Title: Pathway-instructed therapeutic selection of ruxolitinib reduces neuroinflammation in fungal postinfectious inflammatory syndrome.

doi: 10.1126/sciadv.adi9885

Figure Lengend Snippet: Fig. 1. Transcriptional pathway analysis identifies the JAK/STAT inflammatory pathway as predominant in the CSF of patients with cPIIRS. (A to F) Transcriptional analysis of the CFS of patients with cPIIRS (at diagnosis) and non-cPIIRS donor CSF was performed using a NanoString multiplex neuroinflammation panel (n = 4 patients and 3 donors from one experiment). (A) PCA of normalized read counts of 757 total transcripts associated with neuroinflammation for the CSF of patients with cPIIRS and non-cPIIRS donor CSF. (B) Venn diagram indicating the number of DEGs (as described in Methods) in CSF known to be STAT1 (pink) or STAT3 (purple) dependent. Under- lined are the total DEGs for each comparison. (C) Scatterplot of NanoString analysis comparing gene expression profiles of CSF of patients with cPIIRS to non-cPIIRS donor CSF. Highlighted are STAT1 (pink)/STAT3 (purple)–dependent and independent (light blue) DEGs. (D) Top 14 significantly enriched biological pathways identified in the CSF of patients with cPIIRS (MSigDB Hallmark 2020). (E and F) Pathway analysis using DEGs between the CSF of patients with cPIIRS and non-cPIIRS donor CSF was per- formed using Enrichr TRRUST Transcription Factors 2019 Database (E) and the Enrichr Transcription Factor PPI to construct networks (F). (G to I) CSF cells from a patient (at cPIIRS diagnosis, before treatment) were subjected to scRNA-seq. (G) UMAP plot of CSF cell scRNA-seq data showing cell-type clusters used for subsequent analysis (n = 5070). (H) Dotplot depicting selected marker genes in cell clusters. The dot size corresponds to the percentage of cells expressing the gene, and the color indicates the average per cell gene expression. (I) Feature plots of STAT1/STAT3–dependent genes, JAK/STAT genes, and cytokine/cognate cytokine receptor genes using colors to indicate gene expression (log2UMI counts) levels in the UMAP embedding from (G). The number and percent of cells with detected expression for each gene are reported at the bottom of each feature plot.

Article Snippet: RESULTS JAK/STAT pathway prominence in cPIIRS prioritizes the therapeutic candidate to reduce inflammation To identify the optimal therapeutic targets responsible for inflammatory processes in cPIIRS, we evaluated changes in RNA transcripts in cells isolated from the CSF of four patients at the time of cPIIRS diagnosis and three non- cPIIRS donors using a human 757 gene NanoString neuroinflammation multiplex panel (table S1) (11–13).

Techniques: Biomarker Discovery, Multiplex Assay, Comparison, Gene Expression, Construct, Marker, Expressing

Journal: Science advances

Article Title: Pathway-instructed therapeutic selection of ruxolitinib reduces neuroinflammation in fungal postinfectious inflammatory syndrome.

doi: 10.1126/sciadv.adi9885

Figure Lengend Snippet: Fig. 2. Transcriptional pathway analysis identifies the JAK/STAT inflammatory pathway as predominant in a cPIIRS mouse model and disease improvement with ruxolitinib. (A) Graphical schema for the mouse cPIIRS model and daily treatment with ruxolitinib. d, days. (B to G) Gene transcription of mouse brains at 21 dpi was ana- lyzed using a NanoString Neuroinflammation panel (n = 4 mice per group). (B) PCA of mouse brain NanoString results. (C) Venn diagram showing overlapping DEGs in the mouse brain, including STAT1 (pink)–dependent, STAT3 (purple)–dependent, and total (underlined) DEGs. (D) Scatterplots comparing gene expression profiles in mouse brains. Highlighted are STAT1 (pink)– and STAT3 (purple)–dependent DEGs. (E) Top 14 significantly enriched biological pathways identified in the mouse brain (MSigDB Hallmark 2020). (F) Top upstream regulators of DEGs in mouse brains (IPA). Highlighted are JAK/STAT pathway–specific (pink) and consensus downstream JAK/STAT targets (blue). (G) Top IPA-predicted upstream regulators of DEGs in humans (patients with cPIIRS versus non-cPIIRS donors) and mice (Cn infection versus naïve). (H and I) Rux- olitinib concentrations in the mouse brain at 21 dpi (H) and serum (I) (n = 3 mice per group). (J to N) Immunoblot analysis of phospho-STAT1 (Tyr701) [(J) and (K)], phospho- STAT3 (Tyr705) [(J) and (L)], total STAT1 [(J) and (M)], and total STAT3 [(J) and (N)] levels in mouse brains (n = 4 to 8 mice per group; two experiments). (O to T) Brain weight (21 dpi) (O), brain fungal burden (21 dpi) (P), mouse weights (throughout infection) (Q), spleen size (21 dpi) (R), spleen weight (21 dpi) (S), and splenocytes (21 dpi) (T) were enumerated. CFU, colony-forming units. (U) Graphical schema for the mouse cPIIRS model with AMB/ruxolitinib treatment. (V to X) Brain fungal burden (21 dpi) (V), mouse weights (W), and brain weight (21 dpi) (X) were enumerated. Error bars indicate SD from two independent experiments (n = 8 to 20 mice per time point). Student’s t test, with P values indicated above each comparison. Graphical schemas were created with BioRender.com.

Article Snippet: RESULTS JAK/STAT pathway prominence in cPIIRS prioritizes the therapeutic candidate to reduce inflammation To identify the optimal therapeutic targets responsible for inflammatory processes in cPIIRS, we evaluated changes in RNA transcripts in cells isolated from the CSF of four patients at the time of cPIIRS diagnosis and three non- cPIIRS donors using a human 757 gene NanoString neuroinflammation multiplex panel (table S1) (11–13).

Techniques: Gene Expression, Infection, Western Blot, Comparison