Journal: eBioMedicine

Article Title: Multicentre preclinical profiling of apramycin for the treatment of nontuberculous mycobacteria

doi: 10.1016/j.ebiom.2025.106103

Figure Lengend Snippet: Frequency of mutational M. abscessus resistance (FoR) to apramycin (APR) and amikacin (AMK). (a) M. abscessus ATCC 19977. (b) M. abscessus NR-44261. All studies were performed on selective LB agar plates incubated at 37 °C for 5 days. Data are represented as scatter plot, the centre bars represent the geometric mean ( n = 3–4 technical replicates per strain and drug concentration tested). The lower limits of quantification (LoQ) defined by ≤ 1 CFU per plate are indicated by dotted lines. Circular symbols at the LoQ are equivalent to one resistant CFU, triangular symbols pointing downward indicate the absence of resistant CFUs and thus a frequency below that LoQ.

Article Snippet: APR was found to kill intracellular M. abscessus ATCC 19977 (E max = −0.67 log CFU/mL) and intracellular M. avium ATCC 19698 (E max = −1.45 log CFU/mL) in a dose-dependent manner, with the M. avium reference strain (MIC = 4 μg/mL at 37 °C) appearing to be more susceptible to intracellular APR killing than the M. abscessus reference strain (MIC = 0.5 μg/mL at 37 °C) ( d).

Techniques: Incubation, Concentration Assay

Journal: eBioMedicine

Article Title: Multicentre preclinical profiling of apramycin for the treatment of nontuberculous mycobacteria

doi: 10.1016/j.ebiom.2025.106103

Figure Lengend Snippet: Dose-dependent killing of planktonic and intracellular NTMs by apramycin (APR) in comparison to amikacin (AMK). (a) Time- and dose-dependent CFU reduction of M. abscessus reference strains ATCC 19977 (smooth) and NR-44261 (rough) in CAMHB (pH 7.4) determined by sampling, plating, and CFU counting at different time points over 120 h. (b) Luminescence based killing kinetics in standard Middlebrook 7H9 (pH 6.7) and in a 7H9 medium that has been buffered with MES to pH 6.0. (c) Dose–response kill curve for stationary-phase M. abscessus ATCC 19977 in caseum surrogate following five days of drug exposure (mean ± SD; n = 3 technical replicates). (d) Dose response curve of intracellular killing of M. abscessus ATCC 19977 by day 3 and of M. avium ATCC 19698 by day 7 (mean ± SD; n = 3 biological replicates). MXF, moxifloxacin; CLA, clarithromycin.

Article Snippet: APR was found to kill intracellular M. abscessus ATCC 19977 (E max = −0.67 log CFU/mL) and intracellular M. avium ATCC 19698 (E max = −1.45 log CFU/mL) in a dose-dependent manner, with the M. avium reference strain (MIC = 4 μg/mL at 37 °C) appearing to be more susceptible to intracellular APR killing than the M. abscessus reference strain (MIC = 0.5 μg/mL at 37 °C) ( d).

Techniques: Comparison, Sampling

Journal: Science signaling

Article Title: The mechanosensitive channel TRPV4 inhibits pulmonary inflammation by limiting NF-KB signaling in alveolar macrophages

doi: 10.1126/scisignal.adt1539

Figure Lengend Snippet: A. Schematic of P. aeruginosa mouse model with collection of bronchoalveolar lavage (BAL) cells and lung tissue homogenate in alveolar macrophage-specific Trpv4 KO mice ( Cd11c Cre: Trpv4 fl/fl ) and parental controls ( Trpv4 fl/fl ). Created with biorender.com . B. Percent BAL neutrophil, macrophage, and lymphocyte cells counts. n=14–15 mice per group. * p ≤ 0.05, p denotes macrophages in Cd11c Cre: Trpv4 fl/fl mice vs Trpv4 fl/fl mice, ** p ≤0.01, p denotes neutrophils Cd11c Cre: Trpv4 fl/fl vs Trpv4 fl/fl by Student’s t-test. C. CCL3 and CXCL1 chemokine secretion in BAL fluid (BALF) in Trpv4 fl/fl and Cd11cCre: Trpv4 fl/fl by LEGENDplex (pg/mL). n=14–15 mice per group. * p ≤ 0.05, p denotes Cd11c Cre: Trpv4 fl/fl vs Trpv4 fl/fl by Student’s t-test. D. Lung tissue homogenate utilized for flow cytometry with gating on percent CD45 + , F4/80 + , and Siglec-F − population for IL-1β and CCL2 in Cd11c Cre: Trpv4 fl/fl and Trpv4 fl/fl mice as quantified in E. n=14–15 mice per group. * p ≤ 0.05, p denotes IL-1β in Cd11c Cre: Trpv4 fl/fl mice vs Trpv4 fl/fl mice, *** p ≤ 0.001, p denotes CCL2 in Cd11c Cre: Trpv4 fl/fl mice vs Trpv4 fl/fl mice by Student’s t-test.

Article Snippet: Primary antibodies to extracellular and intracellular TRPV4 (Alomone Labs, Jerusalem Israel; Cell Signaling), GAPDH (Fitzgerald Industries International, Acton, MA), total NF-κB/p65 (Cell Signaling), IκBα (Santa Cruz Biotechnology), Integrin β-1 (BD biosciences), β-actin (Abcam), and GSK1016790A (GSK, Sigma-Aldrich) were obtained from commercial vendors.

Techniques: Derivative Assay, Expressing, In Vivo, Flow Cytometry

Journal: Science signaling

Article Title: The mechanosensitive channel TRPV4 inhibits pulmonary inflammation by limiting NF-KB signaling in alveolar macrophages

doi: 10.1126/scisignal.adt1539

Figure Lengend Snippet: A. The mRNA expression of NF-κB genes, including IL - 1β and Cxcl1 , were measured in BMDMs from WT and Trpv4 KO mice ± TLR agonists: TLR1/2 (Pam3CSK4, 100 ng/ml), TLR4 (LPS 100 ng/ml), TLR3 (Poly I:C 10 μg/ml), and TLR9 (CpGDNA 5 μg/ml) at 1 hour. n=3–4 biological replicates per group, *p ≤ 0.05, ** p ≤ 0.01 denotes WT vs KO ± TLR agonist. Student’s t-test comparing Trpv4 KO vs WT BMDMs within a TLR group. B. The same NF-κB genes in response to the TLR agonists were measured at 6 hours. n=3–4 biological replicates per group, *p ≤ 0.05, ** p ≤ 0.01 denotes WT vs KO ± TLR agonist. Student’s t-test comparing Trpv4 KO vs WT BMDMs within a TLR group. C. UMAP cluster plot shows 9 clusters in WT and Trpv4 KO BMDMs ± LPS after 10X genomics. D. Within the Il1β+_Cxcl2+ cluster, the GO BP gene pathway analysis comparing either LPS-stimulated WT or KO represented significant pathways as measured by −Log10 transformed p -value (x-axis; light green: increased KO vs WT, dark green: decreased KO vs WT). n=3 biological replicates per group.

Article Snippet: Primary antibodies to extracellular and intracellular TRPV4 (Alomone Labs, Jerusalem Israel; Cell Signaling), GAPDH (Fitzgerald Industries International, Acton, MA), total NF-κB/p65 (Cell Signaling), IκBα (Santa Cruz Biotechnology), Integrin β-1 (BD biosciences), β-actin (Abcam), and GSK1016790A (GSK, Sigma-Aldrich) were obtained from commercial vendors.

Techniques: Expressing, Transformation Assay

Journal: Science signaling

Article Title: The mechanosensitive channel TRPV4 inhibits pulmonary inflammation by limiting NF-KB signaling in alveolar macrophages

doi: 10.1126/scisignal.adt1539

Figure Lengend Snippet: A. The transcriptional activity of NF-κB response elements was evaluated in NF-κB luciferase transfected HeLa cells, additionally transfected with either empty vector (EV) or TRPV4 plasmid (TRPV4 overexpression) ± LPS (100 ng/mL, 16 hours) and BMDMs from mVenus-RELA (p65) reporter mice transfected with either control (Scrambled) siRNA or TRPV4 siRNA ± LPS (1 μg/mL, 1 hour). HeLa TRPV4 overexpression n=3 biological replicates per group, ****p ≤ 0.0001 , p denotes EV vs TRPV4 expression ± LPS by Student’s t-test and mVenus p65 BMDM. n=5 biological replicates per group, * p ≤ 0.05, ****p ≤ 0.0001 downregulation p denotes Scrambled siRNA vs TRPV4 siRNA ± LPS by Student’s t-test. B. BMDMs from WT and Trpv4 KO mice transfected with control (Scrambled) or p65 siRNA. IL1β secretion was measured by ELISA in the conditional medium from BMDMs treated with LPS (100 ng/mL, 24 hours). n=6 biological replicates per group, Student’s t-test shows no statistical difference. C. BMDMs from WT and Trpv4 KO mice were treated with LPS in the indicated time course. The protein levels of IκBα with representative blot and quantification. n=3 biological replicates per group, * p ≤ 0.05, p denotes IκBα protein WT vs KO at 10 minutes by Student’s t-test. D. BMDMs from WT and Trpv4 KO mice were treated with LPS in the indicated time course. The protein levels of nuclear and cytoplasmic fractions of NF-κB/p65 with representative blot and quantification of nuclear fraction. n=3 biological replicates per group, * p ≤ 0.05, p denotes p65 protein WT vs KO at 30 minutes by Student’s t-test.

Article Snippet: Primary antibodies to extracellular and intracellular TRPV4 (Alomone Labs, Jerusalem Israel; Cell Signaling), GAPDH (Fitzgerald Industries International, Acton, MA), total NF-κB/p65 (Cell Signaling), IκBα (Santa Cruz Biotechnology), Integrin β-1 (BD biosciences), β-actin (Abcam), and GSK1016790A (GSK, Sigma-Aldrich) were obtained from commercial vendors.

Techniques: Activity Assay, Translocation Assay, Luciferase, Transfection, Plasmid Preparation, Over Expression, Control, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Science signaling

Article Title: The mechanosensitive channel TRPV4 inhibits pulmonary inflammation by limiting NF-KB signaling in alveolar macrophages

doi: 10.1126/scisignal.adt1539

Figure Lengend Snippet: A. Sequence alignment of published structure of IκBα (blue) and TRPV4 N-terminal domain shows sequence similarity (grey residues with green selected for structural mapping) within the interacting interface (yellow box and yellow helices) of p65/p50 (red helices) using CLC genomics work bench (Qiagen). Within these homologous regions conserved residues between IκBα and TRPV4 are seen (#1–4, PDB:1NFI). B. 293T cells overexpressing the indicated proteins, Myc-tagged TRPV4 full-length (FL) and Flag-tagged p65, were co-immunoprecipitated for Myc and immunoblotted for Flag and Myc. n=3 biological replicates per group. C. WT BMDMs ± LPS (100 ng/mL, 1 hour) were co-immunoprecipitated for p65 and immunoblotted for p65 and TRPV4, Trpv4 KO as a negative control. n=3 biological replicates per group. D. Trpv4 KO BMDMs were transduced with lentivirus encoding either EV or Myc-tagged TRPV4 full-length ± LPS (100 ng/mL, 1 hour) and stained for Myc (green) and p65 (red); Scale bar: 50 μm, 40X original magnification; magnified cells (inset) shown to see location of TRPV4 and p65 interaction indicated by the yellow color and p65 and nucleus (DAPI) co-localization indicated by the pink color. p65 colocalization with Myc (TRPV4) (at least 25 high power fields were analyzed) and DAPI (40–50 cells were analyzed) was quantified by Pearson’s coefficient. n=3 biological replicates per group. **** p ≤ 0.0001, *** p ≤ 0.001, p denotes Myc-TRPV4 ± LPS by Student’s t-test. E. Schematic demonstrating BiFC method used to determine protein-protein interaction. F. HeLa cells co-transduced with plasmids encoding either empty vector (EV) constructs, VN-tagged TRPV4, or VC-tagged p65. TRPV4 and p65 bind as indicated by VN and VC green fluorescence in the perinuclear region (white arrows); nucleus blue, Scale bar: 75 μm; 40X original magnification; magnified cells (inset) to see location of TRPV4-p65 interaction. Whole image Venus intensity was quantified. n=3 biological replicates per group (at least 25 high power fields were analyzed). ** p ≤ 0.01, p denotes EV vs VN-TRPV4 and VC-p65 by Student’s t-test.

Article Snippet: Primary antibodies to extracellular and intracellular TRPV4 (Alomone Labs, Jerusalem Israel; Cell Signaling), GAPDH (Fitzgerald Industries International, Acton, MA), total NF-κB/p65 (Cell Signaling), IκBα (Santa Cruz Biotechnology), Integrin β-1 (BD biosciences), β-actin (Abcam), and GSK1016790A (GSK, Sigma-Aldrich) were obtained from commercial vendors.

Techniques: Sequencing, Immunoprecipitation, Negative Control, Transduction, Staining, Plasmid Preparation, Construct, Fluorescence

Journal: Science signaling

Article Title: The mechanosensitive channel TRPV4 inhibits pulmonary inflammation by limiting NF-KB signaling in alveolar macrophages

doi: 10.1126/scisignal.adt1539

Figure Lengend Snippet: A. Schematic of NanoBiT constructs with LargeBiT (Lg-BiT) empty vector (green), TRPV4 full length (blue), TRPV4 ANKRD deleted (ANKRD del; burnt orange) and SmallBiT (Sm-BiT) EV (purple) and p65 full length (red). B. 293T cells transfected with plasmids as in A exhibit luminescence with TRPV4 and p65 full length (blue and red stripes) that is decreased with TRPV4 ANKRD del (burnt orange and red stripes) with immunoblot confirmation of transfection efficiency ( Fig. S6 ). n=5 biological replicates per group, **** p ≤ 0.0001, p denotes TRPV4 ± ANKRD deletion by Student’s t-test. C. Trpv4 KO BMDMs transduced with LV as in A exhibit the same luminescence pattern as in B. n=3 biological replicates per group, **** p ≤ 0.0001, p denotes TRPV4 ± ANKRD deletion by Student’s t-test.

Article Snippet: Primary antibodies to extracellular and intracellular TRPV4 (Alomone Labs, Jerusalem Israel; Cell Signaling), GAPDH (Fitzgerald Industries International, Acton, MA), total NF-κB/p65 (Cell Signaling), IκBα (Santa Cruz Biotechnology), Integrin β-1 (BD biosciences), β-actin (Abcam), and GSK1016790A (GSK, Sigma-Aldrich) were obtained from commercial vendors.

Techniques: Binding Assay, Construct, Plasmid Preparation, Transfection, Western Blot, Transduction

Journal: Science signaling

Article Title: The mechanosensitive channel TRPV4 inhibits pulmonary inflammation by limiting NF-KB signaling in alveolar macrophages

doi: 10.1126/scisignal.adt1539

Figure Lengend Snippet: A. WT and Trpv4 KO BMDMs were treated with LPS for the indicated times and cell fractionation of cytosol and endoplasmic reticulum (ER) performed. Quantification of TRPV4 was performed by immunoblot of biochemically isolated cell fractions. n=3 biological replicates per group, * p ≤ 0.05, *** p ≤ 0.001, p denotes ± LPS by One-way ANOVA with Sidak’s MCT. B. Trpv4 KO BMDMs were transduced with Myc-tagged TRPV4 full-length (FL) (stained green) and stimulated with LPS (1 hour). ER was stained with calnexin (red). Co-localization between TRPV4 and calnexin was quantified by Pearson’s coefficient. Data represents scores of Myc-positive individual cells from combined experiments. Scale bar: 50 μm; 40X original magnification; magnified cells (inset) shown to see location of TRPV4 and calnexin interaction. n=3 biological replicates per group (10–40 cells analyzed per experiment), **** p ≤ 0.0001, p denotes ± LPS by Student’s t-test. C. BMDMs from WT mice were treated with LPS for the indicated times, and plasma membrane TRPV4 was measured by immunoblot of biochemically isolated cell fractions. n=3 biological replicates per group, * p ≤ 0.05, p denotes ± LPS by One-way ANOVA with Fischer’s LSD.

Article Snippet: Primary antibodies to extracellular and intracellular TRPV4 (Alomone Labs, Jerusalem Israel; Cell Signaling), GAPDH (Fitzgerald Industries International, Acton, MA), total NF-κB/p65 (Cell Signaling), IκBα (Santa Cruz Biotechnology), Integrin β-1 (BD biosciences), β-actin (Abcam), and GSK1016790A (GSK, Sigma-Aldrich) were obtained from commercial vendors.

Techniques: Clinical Proteomics, Membrane, Cell Fractionation, Western Blot, Isolation, Transduction, Staining

Journal: Science signaling

Article Title: The mechanosensitive channel TRPV4 inhibits pulmonary inflammation by limiting NF-KB signaling in alveolar macrophages

doi: 10.1126/scisignal.adt1539

Figure Lengend Snippet: 1. Under basal conditions, TRPV4 N-terminal tail ANKRD domain interacts with the NF-κB subunit p65, in the complex with IκBα on the endoplasmic reticulum (ER) membrane to limit translocation of p65 to the nucleus for cytokine transcription and secretion. 2. Upon TLR agonism, IκKβ phosphorylates IκBα (3) , which leads IκBα dissociation and proteasome degradation (4). 5. An increased fraction of p65 dissociates from the ER and free p65 translocates to the nucleus thereby limiting pro-inflammatory cytokine transcription and secretion. 6. The dissociated TRPV4 fraction translocates to the plasma membrane. Created with biorender.com .

Article Snippet: Primary antibodies to extracellular and intracellular TRPV4 (Alomone Labs, Jerusalem Israel; Cell Signaling), GAPDH (Fitzgerald Industries International, Acton, MA), total NF-κB/p65 (Cell Signaling), IκBα (Santa Cruz Biotechnology), Integrin β-1 (BD biosciences), β-actin (Abcam), and GSK1016790A (GSK, Sigma-Aldrich) were obtained from commercial vendors.

Techniques: Membrane, Translocation Assay, Clinical Proteomics