Journal: Applied Microbiology and Biotechnology

Article Title: Addition of lactoferrin increases efficacy of three Kayviruses and limits the inflammatory response in pulmonary epithelial cells

doi: 10.1007/s00253-025-13695-9

Figure Lengend Snippet: Cell viability ( A ), LDH release ( B ), and caspase-1 activity ( C ) of the A549 cells treated with phages vB_SauM-A, vB_SauM-C, vB_SauM-D, 1.0 mg/mL lactoferrin, 2.0 mg/mL linezolid, and bacteriophages supplemented with 1 mg/mL lactoferrin in comparison to the untreated negative control and positive control. Arithmetic mean of triplicates with error bars representing SD. Statistical analysis was performed using one-way ANOVA, with *** p < 0.001

Article Snippet: Human carcinoma lung epithelial cell line A549 (ATCC, CCL-185) (ATCC, Manassas, VA, USA) were cultured in 75-cm 2 Culture Flask (Corning, New York, NY, USA) in a F-12 K culture medium (ATCC, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS; ATCC, Manassas, VA, USA), at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 in the HeraCell 150 incubator (Heraeus, Hanau, Germany) in accordance with the supplier’s guidelines.

Techniques: Activity Assay, Comparison, Negative Control, Positive Control

Journal: Applied Microbiology and Biotechnology

Article Title: Addition of lactoferrin increases efficacy of three Kayviruses and limits the inflammatory response in pulmonary epithelial cells

doi: 10.1007/s00253-025-13695-9

Figure Lengend Snippet: The CFU/mL of MRSA strains 70 ( A ), 110 ( B ), and 203 ( C ) in infected A549 cell line after treatment with phages vB_SauM-A, vB_SauM-C, and vB_SauM-D, lactoferrin, linezolid, or phage + Lf combination after 3 h and 6 h post-treatment. Arithmetic mean of triplicates with error bars representing SD. Statistical analysis was performed using the Kruskal–Wallis test followed by Dunn’s multiple comparison test for values with nonparametric distribution, with *** p < 0.001

Article Snippet: Human carcinoma lung epithelial cell line A549 (ATCC, CCL-185) (ATCC, Manassas, VA, USA) were cultured in 75-cm 2 Culture Flask (Corning, New York, NY, USA) in a F-12 K culture medium (ATCC, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS; ATCC, Manassas, VA, USA), at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 in the HeraCell 150 incubator (Heraeus, Hanau, Germany) in accordance with the supplier’s guidelines.

Techniques: Infection, Comparison

Journal: Applied Microbiology and Biotechnology

Article Title: Addition of lactoferrin increases efficacy of three Kayviruses and limits the inflammatory response in pulmonary epithelial cells

doi: 10.1007/s00253-025-13695-9

Figure Lengend Snippet: Cell viability ( A , B , C ) and LDH release ( D , E , F ) from the infected A549 cells treated with phages vB_SauM-A, vB_SauM-C, vB_SauM-D, 1.0 mg/mL lactoferrin, 2.0 mg/mL linezolid, and phage + Lf combination in comparison to the untreated negative control and positive control. Arithmetic mean of triplicates with error bars representing SD. Statistical analysis was performed using one-way ANOVA, with *** p < 0.001, ** p < 0.01, and * p < 0.05

Article Snippet: Human carcinoma lung epithelial cell line A549 (ATCC, CCL-185) (ATCC, Manassas, VA, USA) were cultured in 75-cm 2 Culture Flask (Corning, New York, NY, USA) in a F-12 K culture medium (ATCC, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS; ATCC, Manassas, VA, USA), at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 in the HeraCell 150 incubator (Heraeus, Hanau, Germany) in accordance with the supplier’s guidelines.

Techniques: Infection, Comparison, Negative Control, Positive Control

Journal: Applied Microbiology and Biotechnology

Article Title: Addition of lactoferrin increases efficacy of three Kayviruses and limits the inflammatory response in pulmonary epithelial cells

doi: 10.1007/s00253-025-13695-9

Figure Lengend Snippet: Inflammasome activation measured by caspase-1 activity in the infected A549 cells treated with phages vB_SauM-A, vB_SauM-C, vB_SauM-D, 1.0 mg/mL lactoferrin, 2.0 mg/mL linezolid, and phage + Lf combination in comparison to the untreated negative control and positive control. Arithmetic mean of triplicates with error bars representing SD. Statistical analysis was performed using the Kruskal–Wallis test followed by Dunn’s multiple comparison test for values with nonparametric distribution, with *** p < 0.001

Article Snippet: Human carcinoma lung epithelial cell line A549 (ATCC, CCL-185) (ATCC, Manassas, VA, USA) were cultured in 75-cm 2 Culture Flask (Corning, New York, NY, USA) in a F-12 K culture medium (ATCC, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS; ATCC, Manassas, VA, USA), at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 in the HeraCell 150 incubator (Heraeus, Hanau, Germany) in accordance with the supplier’s guidelines.

Techniques: Activation Assay, Activity Assay, Infection, Comparison, Negative Control, Positive Control

Journal: Nature biotechnology

Article Title: Antimicrobial peptide delivery to lung as peptibody mRNA in anti-inflammatory lipids treats multidrug-resistant bacterial pneumonia

doi: 10.1038/s41587-025-02928-x

Figure Lengend Snippet: a , Relative luminescence intensity of TS LNP-FLuc mRNA in MLG cells. Intensity was normalized to SM-102 LNP group. b , Relative luminescence intensity of TS LNP-FLuc mRNA in A549 cells. Intensity was normalized to SM-102 LNP group. c , Luminescence intensity in the lungs of mice intratracheally injected with TS LNPs-FLuc mRNA. d , Representative IVIS images of lung tissues from c . e , Luminescence intensity in the lungs of mice following IT injection of SM-102 LNP-FLuc mRNA, TS41 LNP-FLuc mRNA, or TS41S LNP-FLuc mRNA. f , Representative IVIS images of lung tissues from e . g , Schematic illustration showing that delivery of Cre recombinase mRNA induces tdTomato expression in Ai14 reporter mice through Cre-mediated recombination. h , Flow cytometry analysis of tdTomato expression in various lung cell types following IT injection of SM-102 LNP-Cre mRNA or TS41S LNP-Cre mRNA. All data are from n = 3 biologically independent samples and are presented as mean values ± s.d. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The murine lung fibroblast MLG cell line (CCL-206), human lung epithelial A549 cell line (CCL-185) and murine macrophage RAW 264.7 cell line (TIB-71) were purchased from the American Type Culture Collection (ATCC).

Techniques: Injection, Expressing, Flow Cytometry

Journal: Nature biotechnology

Article Title: Antimicrobial peptide delivery to lung as peptibody mRNA in anti-inflammatory lipids treats multidrug-resistant bacterial pneumonia

doi: 10.1038/s41587-025-02928-x

Figure Lengend Snippet: a , Table for two rounds of TS41 LNP optimization. Chol, cholesterol . b , Orthogonal assay to evaluate the impact trends of individual lipid component in TS41 LNP formulation at four levels in MLG cells. c , Luminescence intensity fold changes of the two rounds of optimization in MLG cells. d , Orthogonal assay to evaluate the impact trends of individual lipid component in TS41 LNP formulation at four levels in A549 cells. e , Luminescence intensity fold changes of the two rounds of optimization in A549 cells. f , Hydrodynamic diameter and PDI of TS41S LNP. PDI, polydispersity index. g , Zeta potential and encapsulation efficiency of TS41S LNP. h , Representative Cryo-TEM image of TS41S LNP. Scale bar = 100 nm. Data in b-g are from n = 3 biologically independent samples. Data in b and d are plotted as floating bar charts, where each bar represents the range of luminescence intensity from a start value (minimum intensity) to an end value (maximum intensity), with a line indicating the mean intensity. Data in c and e-g are presented as mean values ± SD. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test. ** P < 0.01, **** P < 0.0001.

Article Snippet: The murine lung fibroblast MLG cell line (CCL-206), human lung epithelial A549 cell line (CCL-185) and murine macrophage RAW 264.7 cell line (TIB-71) were purchased from the American Type Culture Collection (ATCC).

Techniques: Formulation, Zeta Potential Analyzer, Encapsulation, Comparison

Journal: The Journal of Biological Chemistry

Article Title: NITAC-mediated ISGylation of eIF4E2 attenuates GSK3β proline-directed kinase activity, conferring cytoprotection

doi: 10.1016/j.jbc.2025.110777

Figure Lengend Snippet: NITAC-enabled activation of eIF4E2 ISGylation. A , schematic diagram of the Nb.30C7-NITAC structure. B and E , Nb.30C7-NITAC enhanced the ISGylation of Flag-eIF4E2. B , A549 or ( E ) HeLa cells were transfected with plasmids expressing FLAG-eIF4E2, HA-Nb.30C7, HA-HECT, HA-Nb.30C7/HA-HECT and HA-Nb.30C7-NITAC as indicated for 12 h and then treated with IFNβ (1000 U/ml) for another 36 h. Then, cell extracts were subjected to Co-IP with anti-ISG15 antibody, followed by IB analysis. C and F , Nb.30C7-NITAC enhances the ISGylation of endogenous eIF4E2. C , A549 or ( F ) HeLa cells were transfected with plasmids expressing HA-Nb.30C7, HA-HECT, HA-Nb.30C7/HA-HECT, and HA-Nb.30C7-NITAC as indicated for 12 h and then treated with IFNβ (1000 U/ml) for another 36 h. Then, cell extracts were subjected to Co-IP with anti-ISG15 antibody, followed by IB analysis. D and G , Nb.30C7-NITAC enhances the ISGylation of exogenous eIF4E2 in a dose-dependent manner. D , A549 or ( G ) HeLa cells were transfected with plasmids (0, 0.2, 0.4, 0.6, or 0.8 μg) expressing HA-Nb.30C7-NITAC for 12 h and then treated with IFNβ (1000 U/ml) for another 36 h. Then, cell extracts were subjected to Co-IP with anti-ISG15 antibody, followed by IB analysis. Co-IP, coimmunoprecipitation; eIF4E2, eukaryotic translation initiation factor 4E2; IB, immunoblot; IFNβ, interferon β; NITAC,Nanobody-based ISGylation Targeting Chimera.

Article Snippet: HEK293T human embryonic kidney cell line, A549 human lung adenocarcinoma epithelial cell line, HeLa human cervical cancer cell line, HCT116 colon cancer cell line, HT22 mouse hippocampal neuronal cell line, and BV2 mouse microglial cell lines were obtained from American Type Culture Collection and China Center for Type Culture Collection.

Techniques: Activation Assay, Transfection, Expressing, Co-Immunoprecipitation Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: NITAC-mediated ISGylation of eIF4E2 attenuates GSK3β proline-directed kinase activity, conferring cytoprotection

doi: 10.1016/j.jbc.2025.110777

Figure Lengend Snippet: ISGylation of eIF4E2 enhances its interaction with GSK3β. A and B , GSK3β interacts with ISGylated eIF4E2. A , A549 cells were transfected with plasmids expressing HA-eIF4E2(WT), HA-eIF4E2(4KR), FLAG-GSK3β, and HERC5 as indicated for 12 h and then treated with IFNβ (1000 U/ml) for another 36 h. Then, cell extracts were subjected to Co-IP with anti-FLAG antibody, followed by IB analysis. ( B ) HEK293T cells cotransfected components of the His 6 -ISGylation system (UBE1L, UBCH8, and His 6 -ISG15), plasmids expressing Myc-eIF4E2(WT/4KR), FLAG-GSK3β, HA-Nb.30C7/HA-HECT, and HA-Nb.30C7-NITAC as indicated for 48 h, and then cell extracts were subjected to Co-IP with anti-FLAG antibody and Ni-NTA pull down to precipitate His 6 -ISG15 and its complexes, followed by IB analysis. C , depletion of ISG15 inhibits GSK3β from interacting with ISGylated eIF4E2. A549 cells were first transfected with siRNA to knock down the ISG15. After 24 h, plasmids expressing FLAG-eIF4E2, HA-GSK3β, and HERC5 were transfected into the cells, and simultaneously, the cells treated with IFNβ (1000 U/ml) for 36 h. Cell extracts were subjected to co-IP with anti-FLAG antibody, followed by IB analysis. D and E , ISGylation of eIF4E2 enhances its interaction with GSK3β. D , HEK293T cells cotransfected components of the His 6 -ISGylation system (UBE1L, UBCH8, and His 6 -ISG15), plasmids expressing FLAG-eIF4E2(WT/4KR) and HERC5 as indicated for 48 h, and then cell extracts were subjected to co-IP with anti-FLAG antibody and Ni-NTA pull down to precipitate His 6 -ISG15 and its complexes, followed by IB analysis. E , A549 cells were transfected with plasmids expressing FLAG-eIF4E2(WT/4KR), HERC5, and HA-Nb.30C7-NITAC as indicated for 12 h and then treated with IFNβ (1000 U/ml) for another 36 h. Then, cell extracts were subjected to co-IP with anti-FLAG antibody, followed by IB analysis. Co-IP, coimmunoprecipitation; eIF4E2, eukaryotic translation initiation factor 4E2; GSK3β, glycogen synthase kinase 3β; IB, immunoblot; IFNβ, interferon β; NITAC, Nanobody-based ISGylation Targeting Chimera.

Article Snippet: HEK293T human embryonic kidney cell line, A549 human lung adenocarcinoma epithelial cell line, HeLa human cervical cancer cell line, HCT116 colon cancer cell line, HT22 mouse hippocampal neuronal cell line, and BV2 mouse microglial cell lines were obtained from American Type Culture Collection and China Center for Type Culture Collection.

Techniques: Transfection, Expressing, Co-Immunoprecipitation Assay, Knockdown, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: NITAC-mediated ISGylation of eIF4E2 attenuates GSK3β proline-directed kinase activity, conferring cytoprotection

doi: 10.1016/j.jbc.2025.110777

Figure Lengend Snippet: ISGylation of eIF4E2 inhibits GSK3β kinase activity. A , ISGylation inhibited the S/T-P phosphorylation of downstream substrates of the eIF4E2–GSK3β pathway. HEK293T cells cotransfected components of the ISGylation system (UBE1L, UBCH8, and ISG15), plasmids expressing FLAG-eIF4E2and HHARI as indicated for 48 h, and then cell extracts were subjected to co-IP with anti-FLAG antibody, followed by IB analysis. B and C , ISGylated eIF4E2 inhibited the S/T-P phosphorylation of downstream substrates of the eIF4E2–GSK3β pathway. B , HCT116 cell transfected plasmids expressing HA-Nb.30C7, HA-HECT, HA-Nb.30C7/HA-HECT, and HA-Nb.30C7-NITAC as indicated for 24 h and treated with CPT (0.5 μM) for another 24 h. Co-IP with anti-ISG15 and IB analysis of whole-cell extracts. C , A549 cell transfected plasmids expressing HA-Nb.30C7, HA-HECT, HA-Nb.30C7/HA-HECT, and HA-Nb.30C7-NITAC as indicated for 12 h and treated with IFNβ (1000 U/ml) for another 36 h. Co-IP with anti-ISG15 and IB analysis of whole-cell extracts. D , ISGylated eIF4E2 inhibits GSK3β′s proline-directed kinase activity. E3 ligase HERC5, E2 conjugating enzyme UBE1L, and UBCH8 protein were synthesized by in vitro transcription and translation. In vitro ISGylation assays were conducted in the presence of ISG15, E1, E2, HERC5, and FLAG-eIF4E2. FLAG-GSK3β, FLAG-eIF4E2, and FLAG-eIF4E2(4KR) proteins were overexpressed and purified from HEK293T cells. His 6 -tagged p53 and ISG15 were expressed and purified from Escherichia coli . eIF4E2, HERC5, UBCH8, UBE1L, and ISG15 were incubated in an ATP-containing buffer at 37 °C for 1 h to induce ISGylation of eIF4E2. After the reaction, the resulting components were combined with GSK3β and p53 in an ATP-containing buffer and incubated at 30 °C for 1 h to perform the in vitro kinase assay, followed by IB analysis. Co-IP, coimmunoprecipitation; CPT, camptothecin; IB, immunoblot; eIF4E2, eukaryotic translation initiation factor 4E2; GSK3β, glycogen synthase kinase 3β; NITAC, Nanobody-based ISGylation Targeting Chimera; S/T-P, proline-directed serine/threonine.

Article Snippet: HEK293T human embryonic kidney cell line, A549 human lung adenocarcinoma epithelial cell line, HeLa human cervical cancer cell line, HCT116 colon cancer cell line, HT22 mouse hippocampal neuronal cell line, and BV2 mouse microglial cell lines were obtained from American Type Culture Collection and China Center for Type Culture Collection.

Techniques: Activity Assay, Phospho-proteomics, Expressing, Co-Immunoprecipitation Assay, Transfection, Synthesized, In Vitro, Purification, Incubation, Kinase Assay, Western Blot