Journal: Science Advances

Article Title: A dynamic gene regulatory code drives synaptic development of hippocampal granule cells

doi: 10.1126/sciadv.adx5140

Figure Lengend Snippet: ( A ) Scheme of the hippocampal formation and situation of GCs in the hippocampal trisynaptic circuit: GCs (red) in the DG receive excitatory inputs from the entorhinal cortex (EC; blue) and send axons to CA3. The inset shows postnatal GC development: NPCs are generated on P1; these will then give rise to NBs and iGCs, which extend dendrites to receive input from mossy cells and send axons toward the CA3 region. At P7, iGCs give rise to mGCs, which receive EC input and develop protrusions along their axon. At P14, mGCs present a complex dendritic arbor, increased dendritic spine density, and complex MFBs along their axon. At P28, mGCs reach peak dendritic spine density, while MFBs are refined, having fewer filopodia. ( B ) Hippocampi of Rbp4-Cre:Ai9 mice at P7, P10, P15, and P28. tdTomato (red) labels GCs in the outer part of the GC layer at each time point. Scale bars, 500 μm. ( C ) Time points used for scRNA-seq experiments; n = 1 mouse per time point, except P28 ( n = 2). ( D ) PCA of highly variable genes (HVGs) separates cells by time point. ( E ) PCA colored by the expression of the GC marker gene calbindin 1 ( Calb1 ). ( F ) Heatmap of the eight clusters obtained from the top 50 DEGs of each pairwise comparison between time points, highlighting, for each cluster, genes present in the SynGO database within the top 20 genes of each pairwise comparison. Cells are ordered by inferred pseudotime, and values represent GAM-fitted expression values for each gene. ( G ) Dot plot representing GO BP terms enriched in each cluster.

Article Snippet: The enzymatic solution was placed in a 37°C water bath while carbogenating and incubated for 10 min (for P5, P7, and P10), 15 min (for P15), and 28 min (for P28), followed by the addition of trypsin (0.125%; Sigma-Aldrich, 59417C) and deoxyribonuclease (DNase I; 0.2 mg/ml; Thermo Fisher Scientific, 10649890) for 2 min (except for the P28 sample, which received no trypsin or DNase I).

Techniques: Generated, Expressing, Marker, Comparison

Journal: Science Advances

Article Title: A dynamic gene regulatory code drives synaptic development of hippocampal granule cells

doi: 10.1126/sciadv.adx5140

Figure Lengend Snippet: ( A ) Hippocampi of Rbp4-Cre:RiboTag mice at P5, P7, P15, and P28. The HA tag (orange) labels the ribosomal subunit RPL22 expressed in GCs in the outer part of the GC layer at all time points. Scale bars, 500 μm. ( B ) Timepoints used for RiboTag experiments; n = 3 or 4 mice for P5 and P7 and n = 2 mice for P10 to P28. ( C ) Methodology for RiboTag experiments. Hippocampi from Rbp4-Cre:RiboTag mice were homogenized, and the homogenate was incubated with anti-HA magnetic beads for RPL22-HA pull-down. Ribosome-bound mRNA was separated from ribosomes, RNA was isolated, and libraries were prepared for bulk RNA-seq. ( D ) PCA shows the separation of biological replicates by time point. ( E ) Heatmap of SCENIC+ eRegulons leading TF (compare with ) expression in RiboTag samples across time points. The label on the left side of the heatmap indicates hierarchical clustering of the eRegulons (see fig. S3G). ( F ) Comparative plots of standardized expression across time for Klf7 , Bcl11b , Bcl6 , and Smad3 show TF expression in snMO (gene expression; light blue line), TF expression in RiboTag (light orange line), mean TG expression in snMO (gene expression; turquoise line), mean TG expression in RiboTag (orange line), and mean TG expression in RiboTag ±1 SD (cream shade around mean TG expression).

Article Snippet: The enzymatic solution was placed in a 37°C water bath while carbogenating and incubated for 10 min (for P5, P7, and P10), 15 min (for P15), and 28 min (for P28), followed by the addition of trypsin (0.125%; Sigma-Aldrich, 59417C) and deoxyribonuclease (DNase I; 0.2 mg/ml; Thermo Fisher Scientific, 10649890) for 2 min (except for the P28 sample, which received no trypsin or DNase I).

Techniques: Incubation, Magnetic Beads, Isolation, RNA Sequencing, Expressing, Gene Expression, Cream

Journal: Science Advances

Article Title: A dynamic gene regulatory code drives synaptic development of hippocampal granule cells

doi: 10.1126/sciadv.adx5140

Figure Lengend Snippet: ( A ) PCA based on eRegulon target gene cell enrichment scores (AUC), colored by Bcl6(+) target gene AUC. ( B ) GO cellular component (CC) terms enriched among Bcl6(+) targets. ( C ) Heatmap of mean normalized accessibility for the 24 predicted Bcl6(+) target regions across time points. ( D ) Coverage plot of the Bcl6 -linked region near Nptx1 [chromosome 11 (chr11): 119550565 and 119551065], with arcs showing SCENIC+-predicted region-gene links. ( E ) AAV vectors used for sparse GC labeling: pAAV-U6-TRE-fDIO-mGFP-IRES-tTA (with scrambled or Bcl6 shRNA) and pAAV-TRE-FLP. IRES, internal ribosomal entry site. FRT (FLP recognition target) sites are indicated in yellow. ( F ) Experimental timeline: P0 DG injections of AAVs in C57Bl6J pups; brains collected at P28 for dendritic and MFB morphological analysis and at P27 to P35 for ephys recordings. ( G ) Representative images of dendritic segments from the medial molecular layer of the DG for control (gray) and Bcl6 KD (green) animals. ( H ) Bcl6 KD reduces spine density and mushroom spine density ( n = 8 mice per group, 20-μm segments per animal; nested t tests, P = 0.0135 and P = 0.0220). ( I ) Representative images of MFBs in control and Bcl6 KD animals. ( J ) Bcl6 KD increases MFB filopodial number ( n = 6 or 7 mice per group, six to eight MFBs per animal; P = 0.0209), with unchanged filopodial length. Box plots show the median, interquartile range, and range (each dot, one animal). ( K ) Representative mEPSC traces from GCs expressing control or Bcl6 shRNAs. ( L ) Bcl6 KD results in reduced mEPSC frequency ( n = 43 or 44 neurons, six mice per group; P = 0.0189, Mann-Whitney test), with unchanged amplitudes. ( M ) Representative mIPSC traces from GCs expressing control or Bcl6 shRNAs. ( N ) Bcl6 KD results in reduced mIPSC frequency ( n = 43 or 44 neurons, six mice per group; P = 0.0101, Mann-Whitney test), with unchanged amplitudes. Individual data points are shown, and bars represent the means ± SEM.

Article Snippet: The enzymatic solution was placed in a 37°C water bath while carbogenating and incubated for 10 min (for P5, P7, and P10), 15 min (for P15), and 28 min (for P28), followed by the addition of trypsin (0.125%; Sigma-Aldrich, 59417C) and deoxyribonuclease (DNase I; 0.2 mg/ml; Thermo Fisher Scientific, 10649890) for 2 min (except for the P28 sample, which received no trypsin or DNase I).

Techniques: Labeling, shRNA, Control, Expressing, MANN-WHITNEY

Journal: Science Advances

Article Title: A dynamic gene regulatory code drives synaptic development of hippocampal granule cells

doi: 10.1126/sciadv.adx5140

Figure Lengend Snippet: ( A ) PCA based on eRegulon target gene cell enrichment scores (AUC) colored by Smad3(+) AUC scores of target genes. ( B ) Heatmap showing mean normalized accessibility per time point of 649 predicted Smad3(+) target regions and labeling region-linked genes within the synaptic organization GO term. ( C ) Coverage plot for the Tanc1 -associated region across time points in mGCs. The target region is located on chr2 between base pairs 59,537,947 and 59,538,447 (gray shade). Arcs represent SCENIC+-predicted region-gene links. ( D ) Plasmids pAAV-U6-TRE-fDIO-mScarlet-IRES-tTA containing control gRNA ( lacZ ) or Smad3 gRNAs and pAAV-TRE-DIO-FLP. FRT sites are indicated with yellow arrowheads; loxP sites (Cre recognition target) are indicated with green arrowheads. ( E ) Experimental design for Smad3 in vivo experiments. Rbp4-Cre:Rosa26-LSL-Cas9 P7 pups were injected in the DG with control or Smad3 gRNA AAVs and AAV-DIO-TRE-FLP for sparse labeling. Morphological analysis of GC dendrites (spine density and morphology) and MFB morphology (area, volume, filopodial number, and length) was performed at P28, and ephys was performed at P27 to P31. ( F ) Representative mEPSC traces from GCs expressing control or Smad3 gRNAs. ( G ) Analyzed mEPSC frequency and amplitude (number of neurons: n = 45 from n = 4 mice for control and n = 42 from n = 4 mice for Smad3 KO conditions; Mann-Whitney test). ( H ) Representative mIPSC traces from GCs expressing control or Smad3 gRNAs. ( I ) Analyzed mIPSC frequency and amplitude (number of neurons: n = 45 from n = 4 mice for control and n = 42 from n = 4 mice for Smad3 KO conditions; Mann-Whitney test, P value = 0.01). Individual data points are shown in (G) and (I), and bars represent the means ± SEM.

Article Snippet: The enzymatic solution was placed in a 37°C water bath while carbogenating and incubated for 10 min (for P5, P7, and P10), 15 min (for P15), and 28 min (for P28), followed by the addition of trypsin (0.125%; Sigma-Aldrich, 59417C) and deoxyribonuclease (DNase I; 0.2 mg/ml; Thermo Fisher Scientific, 10649890) for 2 min (except for the P28 sample, which received no trypsin or DNase I).

Techniques: Labeling, Control, In Vivo, Injection, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Oncolytic HSV-IL27 expression improves CD8 T cell function and therapeutic activity in syngeneic glioma models

doi: 10.1101/2025.05.12.653429

Figure Lengend Snippet: (A) Schematic illustration of C134 and C027. C134 is a second-generation oHSV with deletion of the γ134.5 genes and insertion of the human cytomegalovirus (HCMV) IRS1 gene in the UL3/UL4 intergenic region. C027 was generated from C134 by insertion of bicistronic copies of murine IL-27 (mIL27) in the deleted γ134.5 regions under the MND promoter. (B) mIL27 (p28) secretion from C027 or C134 infected (MOI=1) CT-2A cell supernatants at 24- and 48-hours post-infection measured by ELISA. (C) Viral replication in CT-2A cells infected with C027, C134, or WT HSV (MOI=1) demonstrating viral replication kinetics. Experimental design schematics and representative Kaplan-Meier survival curves in (D) CT-2A (n=11 per cohort), (F) KR158 (n=5- 10 per cohort), and (H) SB28 (n=10 per cohort) immunocompetent syngeneic murine glioma models (Log- Rank test of significance) after treatment with Saline or equivalent PFU (1×10 7 ) of C027 or C134 (parental control). In vivo viral recovery from murine brain tumors (E) CT-2A, (G) KR158, and (I) SB28 at two days post- treatment with C134 (n=3) or C027 (n=3). For B, C, E, G, and I, data are mean ± SEM with each shape representing one replicate or animal. Statistical analyses were performed using two-way analysis of variance with Holm-Šídák’s correction for multiple comparisons (B), unpaired two-tailed Student’s t-test (E, G, and I), or Log-Rank tests of significance (D, F, and H). IC, intracranial; oHSV, oncolytic herpes simplex virus; PFU, plaque forming unit; MOI, multiplicity of infection; IL, interleukin; Tx, treatment.

Article Snippet: Antibodies used for in vivo experiments include: anti-CD8a (2.43, Leinco), anti-IL27 p28 (MM27.7B1, BioXCell), rat IgG2b isotype (LTF-2, BioXCell), mouse IgG2a isotype (N123, Leinco) and were administered by intraperitoneal injection on indicated days.

Techniques: Generated, Infection, Enzyme-linked Immunosorbent Assay, Saline, Control, In Vivo, Two Tailed Test, Virus

Journal: iScience

Article Title: Interferon-γ and IL-27 positively regulate type 1 regulatory T cell development during adaptive tolerance

doi: 10.1016/j.isci.2025.112308

Figure Lengend Snippet:

Article Snippet: Mouse Anti-Mouse IL-27 p28 (clone MM27.7B1) , BioXCell , Cat #BE0326; RRID:AB_2819053.

Techniques: Recombinant, Saline, Staining, Red Blood Cell Lysis, Sequencing, Software

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Flow chart representing the recruitment of LTx patients for analysis of the neutrophil phenotype (cohort 1) and CXCL8 proteoforms (cohort 2). ( A ) To study the neutrophil phenotype, a first cohort of 100 fresh BAL fluid and 50 peripheral blood samples were collected from 134 individual patients, including 16 patients with a paired BAL fluid and peripheral blood sample. BAL fluid samples were excluded for flow cytometry analysis if cell counts were insufficient or upon severe blood contamination. Samples with insufficient neutrophils for proper flow cytometry analysis were excluded as well. Other reasons for exclusion were samples derived from patients on post-operative day 1 or upon hospital discharge immediately after transplantation, from patients with other severe complications (anastomotic stricture, pulmonary embolisms) or without clear diagnosis. Samples were categorized retrospectively to be derived from LTx patients with CLAD or pulmonary infection or stable LTx recipients according to clinical guidelines (see section). ( B ) To study CXCL8 proteoforms in LTx, COVID-19 and influenza patients, -80 °C-stored BAL fluid supernatants from a second cohort of 55 individual patients were used

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Flow Cytometry, Derivative Assay, Transplantation Assay, Biomarker Discovery, Infection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Clinical characteristics CXCL8 proteoform cohort

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Infection, Biomarker Discovery, Cell Counting

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Detection of endogenous CXCL8 proteoforms in BAL fluid. ( A - C ) Neutrophil counts and CXCL8 levels in BAL fluid samples from CLAD ( n = 12), infected ( n = 6) and stable LTx patients ( n = 6) used for CXCL8 proteoform characterization. ( D , E ) Endogenous CXCL8 proteoforms were determined by ISTAMPA in the BAL fluid supernatants of LTx patients with CLAD or infection. Total CXCL8 levels were below the detection limit for ISTAMPA analysis in stable LTx recipients. CXCL8 proteoforms were presented according to their relative abundance in the BAL fluids, expressed as percentage of total CXCL8. ( F , G ) Correlation between the relative abundance of the most potent proteoform CXCL8(9–77) and the percentage and absolute numbers of neutrophils in the LTx BAL fluid samples (CLAD and infection combined). ( H , I ) ISTAMPA analysis of endogenous CXCL8 proteoforms in COVID-19 ( n = 24) and influenza ( n = 7) BAL fluid supernatants. Data are shown as box-and-whisker plots (box: median with interquartile range, whiskers: full data distribution), with each dot representing an individual BAL fluid sample, and statistically analyzed by Kruskal-Wallis tests with Dunn’s multiple comparisons tests: * p < 0.05; ** p < 0.01; *** p < 0.001. Correlation analysis was performed calculating a Spearman correlation coefficient and plotted utilizing a simple linear regression line

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Infection, Whisker Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Proteolytic processing of intact CXCL8 in BAL fluid. ( A ) Recombinant human CXCL8(1–77) [125 ng] was spiked in 10 µL of BAL fluid supernatant from CLAD ( n = 12), infected ( n = 6) or stable LTx patients ( n = 6) and from patients with COVID-19 ( n = 10) or influenza ( n = 7). After incubation for 3 h at 37 °C, CXCL8 proteolysis was analyzed by ISTAMPA. ( B ) CXCL8 proteoforms were also determined immediately after spiking CXCL8 [without 37 °C incubation but including the required 30 min pre-purification of the CXCL8 proteoforms at room temperature (RT) as part of the ISTAMPA procedure] and after 16 h of incubation at 37 °C in the COVID-19 BAL fluids. CXCL8 proteoforms were presented according to their relative abundance in the BAL fluids, expressed as percentage of total CXCL8. ( C , D ) Correlation between the relative abundance of the intact CXCL8(1–77) or the truncated CXCL8(6–77) proteoform after 3 h incubation at 37 °C and the elastinolytic activity in the COVID-19 BAL fluid samples . Data are shown as box-and-whisker plots (box: median with interquartile range, whiskers: full data distribution), with each dot representing an individual BAL fluid sample, and statistically analyzed by Kruskal-Wallis tests with Dunn’s multiple comparisons tests: * p < 0.05; **** p < 0.0001. Correlation analysis was performed calculating a Spearman correlation coefficient and plotted utilizing a simple linear regression line

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Recombinant, Infection, Incubation, Purification, Activity Assay, Whisker Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Inhibition of proteolytic processing of CXCL8 in BAL fluid. ( A ) Recombinant human CXCL8(6–77) [125 ng] was spiked in 10 µL of BAL fluid supernatant ( n = 3–6) from COVID-19 (indicated by dots) and CLAD patients (indicated by triangles) and incubated for 3 h at 37 °C, in the absence or presence of the metalloprotease inhibitor EDTA. ( B ) Recombinant human CXCL8(1–77) [125 ng] was spiked in 10 µL of BAL fluid supernatant ( n = 8–9) from COVID-19 (dots) and CLAD patients (triangles) and incubated for 3 h at 37 °C, in the absence or presence of the serine protease inhibitor AEBSF or/and the metalloprotease inhibitor EDTA. After incubation, CXCL8 proteolysis was determined by ISTAMPA. All CXCL8 proteoforms were presented according to their relative abundance in the sample, expressed as percentage of total CXCL8. Data are shown as box-and-whisker plots (box: median with interquartile range, whiskers: full data distribution), with each dot representing an individual BAL fluid sample, and statistically analyzed by Mann-Whitney U tests or Kruskal-Wallis tests with Dunn’s multiple comparisons tests: * p < 0.05; *** p < 0.001; **** p < 0.0001

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Inhibition, Recombinant, Incubation, Protease Inhibitor, Whisker Assay, MANN-WHITNEY