Journal: Molecular Genetics and Metabolism Reports

Article Title: Empagliflozin in GSD-Ib: Long-term safety and sustained recovery of neutrophil function including NET formation

doi: 10.1016/j.ymgmr.2026.101345

Figure Lengend Snippet: Empagliflozin restores neutrophil function in a GSD-Ib infant (P1) by lowering 1,5-AG and enhancing cell survival, NET formation, ROS activity, and bacterial killing. Highly purified human blood neutrophils were isolated from healthy donors and from P1, both before and after empagliflozin (empa) treatment. If required for the experiments, cells were primed with GM-CSF for 20 min and subsequently activated with 10 −8 M C5a, or stimulated with 25 nM PMA without prior priming. (A) Neutrophil viability. Cell death was assessed by propidium iodide (PI) uptake at the indicated time points ( n = 7). (B) Plasma concentrations of 1,5-AG were measured using liquid chromatography mass spectrometry (LC-MS). Box plots indicate the 5th–95th percentile. Differences between groups were assessed using the Mann-Whitney U test. (C) NET formation was assessed by confocal microscopy. Extracellular DNA fibers were stained with MitoSOX Red, and nuclei were counterstained with Hoechst 33342, followed by analysis using confocal microscopy ( n = 8). Scale bars, 10 μm. (D) Cells releasing DNA fibers were quantified in 10 randomly selected images for each condition (n = 8). (E) Quantification of dsDNA in supernatants of activated neutrophils using PicoGreen fluorescent dye ( n = 5). (F) Total ROS activity was measured by flow cytometry (n = 8). (G) Bacterial killing of opsonized GFP- E.coli was assessed by flow cytometry. After incubation, supernatants were collected, and the reduction in viable bacteria was determined (n = 7). All data are presented as means ± SEM, with individual measurements depicted as dots. Difference between groups were assessed using unpaired t -test, unless stated otherwise. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant. 1,5-AG, 1,5-anhydroglucitol.

Article Snippet: Human C5a was purchased from Hycult Biotech (Uden, The Netherlands).

Techniques: Activity Assay, Purification, Isolation, Clinical Proteomics, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, MANN-WHITNEY, Confocal Microscopy, Staining, Flow Cytometry, Incubation, Bacteria

Journal: Molecular Genetics and Metabolism Reports

Article Title: Empagliflozin in GSD-Ib: Long-term safety and sustained recovery of neutrophil function including NET formation

doi: 10.1016/j.ymgmr.2026.101345

Figure Lengend Snippet: Marked neutrophil dysfunction and elevated cell death in an untreated GSD-Ib patient (P2). Highly purified human blood neutrophils were isolated from healthy donors and from P2, who was not receiving empagliflozin treatment. When required for the experiments, cells were primed with GM-CSF for 20 min and subsequently activated with 10 −8 M C5a, or stimulated with 25 nM PMA without prior priming. Experimental conditions and stimulation protocols were as described in . (A) Neutrophil viability assessed by propidium iodide uptake. (B) Plasma 1,5-AG concentrations measured by LC-MS. (C) Representative confocal microscopy images of NET formation. Scale bars, 10 μm. (D) Quantification of DNA-releasing neutrophils. (E) Quantification of dsDNA release in culture supernatants. (F) Total ROS activity measured by flow cytometry. (G) Bacterial killing of opsonized GFP- E. coli assessed by flow cytometry. Data are presented as means ± SEM, with individual measurements depicted as dots. Difference between groups were assessed using unpaired t -test, unless stated otherwise. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant. 1,5-AG, 1,5-anhydroglucitol.

Article Snippet: Human C5a was purchased from Hycult Biotech (Uden, The Netherlands).

Techniques: Purification, Isolation, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Confocal Microscopy, Activity Assay, Flow Cytometry

Journal: Journal of Clinical and Translational Hepatology

Article Title: Single-cell Sequencing Reveals Neutrophil Extracellular Traps in Association with Endotheliopathy and Immunothrombosis in Hepatitis B Virus-related Acute-on-chronic Liver Failure

doi: 10.14218/JCTH.2025.00666

Figure Lengend Snippet: (A) Visualization of differentially expressed genes and pathways in neutrophils from HBV-ACLF patients and HC using the circlize R package. (B) mRNA expression levels of TLR2 and C5aR1 in neutrophils from HC, CHB, and HBV-ACLF patients. (C) Immunofluorescence images (SYTOX Green and DAPI) in neutrophils stimulated with plasma from HC, CHB, and HBV-ACLF groups. Scale bars: 50 µm. (D) Quantification of NETs release from neutrophils stimulated with plasma from HC, CHB, and HBV-ACLF groups, measured by SYTOX Green fluorescence. (E) NETs release in the presence or absence of TLR2 or C5aR1 inhibitors. * P < 0.05, *** P < 0.001, and **** P < 0.0001. NETs, neutrophil extracellular traps; HBV-ACLF, hepatitis B virus-related acute-on-chronic liver failure; TLR2, toll-like receptor 2; C5aR1, complement component 5a receptor 1; CHB, chronic hepatitis B; HC, healthy controls.

Article Snippet: In selected experiments, neutrophils were preincubated with the complement component 5a receptor 1 (C5aR1) antagonist (HY-163379; MedChemExpress) or the toll-like receptor 2 (TLR2) antagonist (HY-116213; MedChemExpress) for 30 min prior to plasma stimulation.

Techniques: Expressing, Immunofluorescence, Clinical Proteomics, Fluorescence, Virus

Journal: bioRxiv

Article Title: SARM1 is required for macrophage immunophenotype switching that is essential for nerve repair

doi: 10.64898/2026.04.07.716973

Figure Lengend Snippet: ( A ) Representative still images from live cell imaging sequences following a 2D scratch assay of WT and Sarm1-/- stimulated Mɸ. t=0 is immediately after the scratch and t=48 hours is 48 hours post-scratch. Scratch boundaries are indicated by yellow dashed lines. Scale bar = 300µm. ( B,C ) Quantification of the relative wound density of WT ( B ) or Sarm1-/- ( C ) Mɸ cultures after the scratch assay in A represented as a percentage of t=0. N=3 biological replicates. Error = SEM. *p<0.05; **p<0.01; ***p<0.005; letters (a,b,c) p<0.05. by two-way repeated measures ANOVA with Dunnett post-hoc test. Blue asterisk = mCSF compared to t=0. Green asterisk = IL-4 compared to t=0. Orange asterisk = LPS compared to t=0. a = mCSF compared to IL-4; b = mCSF compared to LPS; c = mCSF compared to IFNɣ conditions at indicated timepoints. ( D ) Representative still images from live cell imaging sequences following a 3D invasion assay in Matrigel of WT and Sarm1-/- stimulated Mɸ. t=0 immediately after the scratch and t=48 hours is 48 hours post-scratch. Scratch boundaries are indicated by yellow dashed lines. Scale bar = 300µm. ( E ) Quantification of the relative wound density of the invasion assay without C5a added (top) and with C5a added as a chemoattractant (bottom) represented as a percentage of t=0. N=2-4 biological replicates. Error = SEM. Letters (b,c) p<0.05; **p<0.01; ***p<0.005; ****p<0.001 by two-way ANOVA, main effects model with Tukey post-hoc test after simple linear regression model indicated slopes were not different. Wildtype Mɸ + C5a main effects could not be calculated because slopes were significantly different. Orange asterisk = LPS compared to t=0. b = mCSF compared to LPS; c = mCSF compared to IFNɣ conditions at indicated timepoints. ( F ) Representative images of F4/80 (green) positive Mɸ stained for Oil Red O lipids (magenta) following a myelin challenge assay. No clearance indicates a 24 hour myelin treatment and clearance indicates 24 hours of myelin treatment followed by myelin removal for 24 hours. Scale bar = 100µm. ( G ) Quantification of OilRedO intensity per Mɸ (normalized to Mɸ cell area). N = 3 biological replicates and each data point = 1 Mɸ. Mean +/- SEM is shown. Outliers were removed with ROUT Q=1. **p<0.01; ***p<0.005; ****p<0.001 by Kruskal-Wallis test with Dunn’s correction for multiple comparisons. Black asterisk = WT to Sarm1-/- comparisons. Purple asterisk = Sarm1-/- to Sarm1-/- comparisons. Blue asterisk = WT to WT comparisons.

Article Snippet: Additional media with or without recombinant mouse complement component C5a (R&D system, #2150-C5/CF) was added after incubation.

Techniques: Live Cell Imaging, Wound Healing Assay, Invasion Assay, Staining

Journal: medRxiv

Article Title: Differential virulence potential of different clades of multidrug-resistant Klebsiella pneumoniae ST258

doi: 10.64898/2026.03.28.26349612

Figure Lengend Snippet: (A and B) Bacteria recovered after 30-minute exposure of 10 8 CFU/mL to 85% human serum. Limit of detection (LOD) is indicated with a dashed line. (A) 13 Clade 1 isolates and 36 Clade 2 isolates were tested, each with at least two biological replicates. (B) 6 biological replicates per strain were tested. (C and D) C3 binding to bacteria was measured using an indirect ELISA. (C) 13 Clade 1 isolates and 36 Clade 2 isolates were tested, each with at least two biological replicates. (D) 6 biological replicates were tested per strain, each with at least 5 technical replicates. (E) Correlation between C3 binding and serum survival of Clade 2 isolates was assessed using Spearman correlation. (F) Uronic acid was quantified from a selection of Clade 1 (n=6) and Clade 2 (n=10) isolates with 3 biological replicates per isolate. Significance was assessed with Welch’s t-test. (G) Correlation between uronic acid quantity and serum survival of 10 Clade 2 isolates was assessed using Spearman correlation. (H) OD-normalized bacteria were reacted with 5% human serum in PBS for 30 minutes. C5a from the supernatant of the reaction was quantified using a sandwich ELISA. At least two biological replicates per isolate were performed. For panels A-D and H, P ≤ 0.05: *, P ≤ 0.01: **, P ≤ 0.001: *** by Mann-Whitney test.

Article Snippet: C5a quantification was performed using the Human Complement Component C5a DuoSet ELISA from R&D systems.

Techniques: Bacteria, Binding Assay, Indirect ELISA, Selection, Sandwich ELISA, MANN-WHITNEY