Journal: bioRxiv

Article Title: Powassan Virus NS5 Antagonizes TYK2-Mediated Immune Signaling Pathways

doi: 10.64898/2026.09.07.749923

Figure Lengend Snippet: ( A ) Schematic depicting TYK2 involvement in IFN-λ and IL-12 signaling. ( B ) HepG2 hepatocyte cells were stimulated with IFN-λ 1 (50ng/mL) for 24hr, infected with POWV SP (MOI 1 for 48hr), then subjected to RNA extraction and qRT-PCR for POWV SP NS5 RNA normalized to 18S RNA ( n =3 independent experiments). ( C, D ) HepG2 hepatoma cells ( C ) or HaCaT keratinocyte cells ( D ) stably expressing POWV NS5 or empty vector controls left unstimulated or stimulated with IFN-λ 1 (50ng/mL) for 24hr, then subjected to RNA extraction and qRT-PCR. Ifit1 expression was normalized to 18S RNA ( n =3 independent experiments; ordinary one-way ANOVA with Šidák’s multiple comparisons test). ( E ) HMC3 cells were infected with POWV SP (MOI 0.1 for 48h), left unstimulated or stimulated with 50ng/mL IFN-λ 1 or 5ng/ml IFN-β for 20min, then lysates were subjected to western blotting. ( F ) HEK-Blue IL-12 cells were infected with POWV SP (MOI 1 for 48h), left unstimulated or stimulated with IL-12 (100ng/mL) for 24hr, and SEAP reporter enzyme activity measured in the supernatant (n=3 independent experiments; Mann-Whitney test). ( G ) HEK-Blue IL-12 cells stably expressing an empty vector control were stimulated with increasing concentrations of IL-12 for 24h and the reporter enzyme activity was measured in the supernatant (n=3 independent experiments). ( H ) HEK-Blue IL-12 cells stably expressing WNV NS5, POWV LB NS5, POWV SP NS5 or an empty vector control were left unstimulated or stimulated with IL-12 (100ng/mL) for 24hr, and the reporter enzyme activity was measured in the supernatant (n=3 independent experiments; Brown-Forsythe ANOVA with Dunnett’s T3 multiple comparisons test). For all data, the mean ± SD is shown. Significance is indicated by ∗∗ p < 0.005.

Article Snippet: HEK-Blue IL-12 cells (Invivogen hkb-il12) were cultured according to manufacturer’s instructions.

Techniques: Infection, RNA Extraction, Quantitative RT-PCR, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Activity Assay, MANN-WHITNEY, Control

Journal: bioRxiv

Article Title: Powassan Virus NS5 Antagonizes TYK2-Mediated Immune Signaling Pathways

doi: 10.64898/2026.09.07.749923

Figure Lengend Snippet: Lysates from HepG2 ( A ) and HaCaT ( B ) cells transduced with lentiviruses to stably express Strep-tagged POWV LB NS5, POWV SP NS5, or empty vector were analyzed via western blot with anti-Strep antibody. ( C ) HEK-Blue IL-12 cells were infected with POWV SP (MOI 1 for 48hr) and viral RNA measured by qRT-PCR. The mean ± SD is shown. ( D ) HEK-Blue IL-12 cells transduced with lentiviruses to stably express Strep-tagged POWV LB NS5, POWV SP NS5, WNV NS5, or empty vector were analyzed via western blot with anti-Strep antibody. Representative images shown.

Article Snippet: HEK-Blue IL-12 cells (Invivogen hkb-il12) were cultured according to manufacturer’s instructions.

Techniques: Transduction, Stable Transfection, Plasmid Preparation, Western Blot, Infection, Quantitative RT-PCR

Journal: PLOS One

Article Title: Activation of conventional and plasmacytoid dendritic cells and enhancement of anti-viral immune responses by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059

doi: 10.1371/journal.pone.0359545

Figure Lengend Snippet: PBMCs are cultured with or without L . bulgaricus OLL1073R-1 or S. thermophilus OLS3059 for 24 h. The expression of CD86, CD40, and HLA-DR in (A) cDCs and (B) pDCs was analyzed by flow cytometry. Representative histograms (black, control; red, OLL1073R-1; blue, OLS3059) and the corresponding geometric mean fluorescent intensity (gMFI) are shown (n = 6). (C) Representative plots of IL-12 expression in cDCs were shown, and the ratio of IL-12 expressing cells in cDCs were analyzed (n = 5). *: p < 0.05, **: p < 0.01, NS: not significant by Dunnett’s test.

Article Snippet: IL-12 levels in the culture supernatants were quantified using an IL-12 by AuthentiKine IL-12 p40 ELISA Kit (Proteintech, Rosemont, USA).

Techniques: Cell Culture, Expressing, Flow Cytometry, Control

Journal: PLOS One

Article Title: Activation of conventional and plasmacytoid dendritic cells and enhancement of anti-viral immune responses by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059

doi: 10.1371/journal.pone.0359545

Figure Lengend Snippet: (A) Isolated cDCs and pDCs are cultured with or without L . bulgaricus OLL1073R-1 for 24 h. IL-12p40, IFN-α2a, IFN-β levels in supernatants were analyzed (n = 3). (B) PBMCs (n = 4), cDCs-depleted PBMCs (n = 3) and pDCs-depleted PBMCs (n = 3) were cultured with or without L . bulgaricus OLL1073R-1 for 24 h. IFN-α2a, IFN-β levels in supernatants were analyzed. (C) PBMCs, cDCs-depleted PBMCs and pDCs-depleted PBMCs were cultured with or without L . bulgaricus OLL1073R-1 under the stimulation of influenza antigen. The ratio of IFN-γ expressing cells in CD4(+) T EM were analyzed 24 h after incubation (n = 5, *: p < 0.05, NS: not significant by Student’s t-test). IL-12 levels in supernatants were analyzed 20 h after incubation (n = 3). *: p < 0.05, NS: not significant by Mann-Whitney U test unless otherwise noted. Data below detection limit were calculated as zero.

Article Snippet: IL-12 levels in the culture supernatants were quantified using an IL-12 by AuthentiKine IL-12 p40 ELISA Kit (Proteintech, Rosemont, USA).

Techniques: Isolation, Cell Culture, Expressing, Incubation, MANN-WHITNEY

Journal: PLOS One

Article Title: Activation of conventional and plasmacytoid dendritic cells and enhancement of anti-viral immune responses by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059

doi: 10.1371/journal.pone.0359545

Figure Lengend Snippet: PBMCs are cultured with multiple L. bulgaricus strains for 24 h. (A) The ratio of IL-12 expressing cells in cDCs and (B) Geometric mean fluorescent intensity (gMFI) of CD86 expression were analyzed (n = 5). **: p < 0.01, NS: not significant by Dunnett’s test.

Article Snippet: IL-12 levels in the culture supernatants were quantified using an IL-12 by AuthentiKine IL-12 p40 ELISA Kit (Proteintech, Rosemont, USA).

Techniques: Cell Culture, Expressing

Journal: PLOS One

Article Title: Activation of conventional and plasmacytoid dendritic cells and enhancement of anti-viral immune responses by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059

doi: 10.1371/journal.pone.0359545

Figure Lengend Snippet: PBMCs are cultured with or without L . bulgaricus OLL1073R-1 or S. thermophilus OLS3059 for 24 h. The expression of CD86, CD40, and HLA-DR in (A) cDCs and (B) pDCs was analyzed by flow cytometry. Representative histograms (black, control; red, OLL1073R-1; blue, OLS3059) and the corresponding geometric mean fluorescent intensity (gMFI) are shown (n = 6). (C) Representative plots of IL-12 expression in cDCs were shown, and the ratio of IL-12 expressing cells in cDCs were analyzed (n = 5). *: p < 0.05, **: p < 0.01, NS: not significant by Dunnett’s test.

Article Snippet: IL-12 levels in the culture supernatants were quantified using an IL-12 by AuthentiKine IL-12 p40 ELISA Kit (Proteintech, Rosemont, USA).

Techniques: Cell Culture, Expressing, Flow Cytometry, Control

Journal: PLOS One

Article Title: Activation of conventional and plasmacytoid dendritic cells and enhancement of anti-viral immune responses by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059

doi: 10.1371/journal.pone.0359545

Figure Lengend Snippet: (A) Isolated cDCs and pDCs are cultured with or without L . bulgaricus OLL1073R-1 for 24 h. IL-12p40, IFN-α2a, IFN-β levels in supernatants were analyzed (n = 3). (B) PBMCs (n = 4), cDCs-depleted PBMCs (n = 3) and pDCs-depleted PBMCs (n = 3) were cultured with or without L . bulgaricus OLL1073R-1 for 24 h. IFN-α2a, IFN-β levels in supernatants were analyzed. (C) PBMCs, cDCs-depleted PBMCs and pDCs-depleted PBMCs were cultured with or without L . bulgaricus OLL1073R-1 under the stimulation of influenza antigen. The ratio of IFN-γ expressing cells in CD4(+) T EM were analyzed 24 h after incubation (n = 5, *: p < 0.05, NS: not significant by Student’s t-test). IL-12 levels in supernatants were analyzed 20 h after incubation (n = 3). *: p < 0.05, NS: not significant by Mann-Whitney U test unless otherwise noted. Data below detection limit were calculated as zero.

Article Snippet: IL-12 levels in the culture supernatants were quantified using an IL-12 by AuthentiKine IL-12 p40 ELISA Kit (Proteintech, Rosemont, USA).

Techniques: Isolation, Cell Culture, Expressing, Incubation, MANN-WHITNEY

Journal: PLOS One

Article Title: Activation of conventional and plasmacytoid dendritic cells and enhancement of anti-viral immune responses by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059

doi: 10.1371/journal.pone.0359545

Figure Lengend Snippet: PBMCs are cultured with multiple L. bulgaricus strains for 24 h. (A) The ratio of IL-12 expressing cells in cDCs and (B) Geometric mean fluorescent intensity (gMFI) of CD86 expression were analyzed (n = 5). **: p < 0.01, NS: not significant by Dunnett’s test.

Article Snippet: IL-12 levels in the culture supernatants were quantified using an IL-12 by AuthentiKine IL-12 p40 ELISA Kit (Proteintech, Rosemont, USA).

Techniques: Cell Culture, Expressing

Journal: ACS Nano

Article Title: Polymer Molecular Weight Influences Cancer Cell Surface Retention and Cytokine Presentation by Layer-by-Layer Nanoparticles

doi: 10.1021/acsnano.6c02817

Figure Lengend Snippet: Assembly of the LbL film under mild ionic strength generates thicker layers but reduces IL-12 bioavailability and limits lipid release. (a) Schematic of LbL film conformation on IL-12 LbL-NPs with assembly in DI water or a higher ionic strength buffer. Negative-stain TEM of IL-12 LbL-NPs assembled either in (b) DI or (c) hNa. Scale bars represent 100 nm. (d) Assessment of LbL-NP film stability via dynamic light scattering measurements (mean ± s.d.) of NP aggregation through exposure of NPs to increasing concentrations of sodium chloride (NaCl). (e) Fluorescently labeled NPs were dosed to HM-1 cells in vitro . Four hours after dosing, cells were washed, and the NP fluorescence associated with cells was measured on a fluorescence plate reader. Shown are the normalized fluorescence readings relative to an unlayered negatively charged liposome (mean ± s.e.m.). (f, g) Fluorescently labeled NPs (cyan) were dosed to HM-1 cells in vitro . Four hours after dosing, cells were washed, fixed, and stained with wheat germ agglutinin (cell membrane stain, green) and Hoechst 33342 (nucleus stain, blue), then visualized via confocal microscopy. Shown are representative confocal microscopy images of HM-1 cells incubated for 4 h with LbL-NPs assembled in (f) DI or (g) hNa. (h) Quantification of fluorescently labeled PLE released upon particle incubation in serum-containing physiological buffer (mean ± s.d.). (i) Quantification of lipid release from LbL-hNa relative to LbL-DI after 24 h incubation in serum-containing media (mean ± s.e.m.). (j–l) B6C3F1 mice ( n = 7/group) inoculated with 10 6 HM-1-luc tumor cells on day 0 were treated on day 7 with 20 μg of IL-12 conjugated to LbL-NPs assembled in either DI or hNa. Shown are (j) the experimental timeline, (k) in vivo IVIS whole animal i.p. BLI readings (mean ± s.e.m.), and (l) overall survival. Statistical comparisons performed using (e) one-way ANOVA with Tukey’s multiple comparisons test, (i) unpaired t -test, (k) unpaired t -test on AUC, and (l) log-rank (Mantel–Cox) test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: HEK-Blue IL-12 (InvivoGen) cells were cultured and used for IL-12 bioactivity assessment according to the manufacturer’s instructions.

Techniques: Staining, Stability, Dynamic Light Scattering Assay, Labeling, In Vitro, Fluorescence, Membrane, Confocal, Microscopy, Incubation, In Vivo

Journal: ACS Nano

Article Title: Polymer Molecular Weight Influences Cancer Cell Surface Retention and Cytokine Presentation by Layer-by-Layer Nanoparticles

doi: 10.1021/acsnano.6c02817

Figure Lengend Snippet: High MW polyelectrolytes stabilize LbL-NP but maintain IL-12 bioactivity and lipid release properties. (a) Schematic IL-12 LbL-NPs with films composed of low- or high-molecular-weight (MW) polymers. (b) Dynamic light scattering size and polydispersity measurements of LbL-NPs composed of LMW or HMW polymers (mean ± s.d.). (c) Apparent surface ζ-potential measurements of LbL-NPs composed of LMW or HMW polymers (mean ± s.d.). (d) Assessment of the LbL film and NP stability via a salt stress test (mean ± s.d.). (e) Release of PLE from LbL-NPs in 10% serum (mean ± s.d.). (f) HEK-Blue IL-12 response (mean ± s.e.m.). (g) Fraction of fluorescently tagged lipid released from outer leaflet in 100% serum from LbL-NPs assembled with low molecular weight polymers either in DI or hNa or high molecular weight polymers assembled in DI (mean ± s.d.). Statistical comparisons performed using (e) two-way ANOVA with Tukey’s multiple comparisons test (**** p < 0.0001).

Article Snippet: HEK-Blue IL-12 (InvivoGen) cells were cultured and used for IL-12 bioactivity assessment according to the manufacturer’s instructions.

Techniques: High Molecular Weight, Polymers, Dynamic Light Scattering Assay, Stability, Molecular Weight, Zeta Potential Analyzer

Journal: ACS Nano

Article Title: Polymer Molecular Weight Influences Cancer Cell Surface Retention and Cytokine Presentation by Layer-by-Layer Nanoparticles

doi: 10.1021/acsnano.6c02817

Figure Lengend Snippet: High MW polyelectrolytes increase cancer cell binding, and increased PLE MW augments the efficiency of the surface presentation of LbL-NPs. (a–d) Fluorescently labeled NPs were dosed to HM-1 cells in vitro . Four hours after dosing, cells were washed and stained with an anti-IL-12 monoclonal antibody. After anti-IL-12 Ab binding, cells were again washed, and the NP and IL-12 fluorescence associated with cells was measured on a fluorescence plate reader. Shown are (a) the normalized fluorescence readings relative to an unlayered negatively charged liposome (mean ± s.e.m.), (b) schematic for staining extracellularly presented IL-12 NPs, (c) the ratio of IL-12 to NP fluorescence of UL and PLR 50 PLE 100 NPs normalized to PLR 50 PLE 100 (mean ± s.e.m.), and (d) the ratio of IL-12 to NP fluorescence for the library of polyelectrolyte MWs normalized to PLR 50 PLE 100 (mean ± s.e.m.). (e, f) Fluorescently labeled NPs were dosed to HM-1 cells in vitro . Four hours after dosing, cells were washed, suspended, stained with an anti-IL-12 monoclonal antibody, rewashed, and fixed. Shown are (e) representative flow plots of IL-12 fluorescence and NP fluorescence of HM-1 cells dosed with 15 μg/mL of PLR 50 PLE 100 (LMW) or PLR 700 PLE 800 (HMW), and (f) quantitation of extracellular IL-12 median fluorescence intensity (MFI) of HM-1 cells dosed with LMW and HMW LbL-NPs across various NP concentrations (mean ± s.d.). Statistical comparisons performed using (a) one-way ANOVA with Tukey’s multiple comparisons test, (c) unpaired t -test, and (d) one-way ANOVA with Tukey’s multiple comparisons test, only shown within PLR groups (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: HEK-Blue IL-12 (InvivoGen) cells were cultured and used for IL-12 bioactivity assessment according to the manufacturer’s instructions.

Techniques: Binding Assay, Labeling, In Vitro, Staining, Fluorescence, Quantitation Assay, Enzyme-linked Immunosorbent Assay, FACS

Journal: ACS Nano

Article Title: Polymer Molecular Weight Influences Cancer Cell Surface Retention and Cytokine Presentation by Layer-by-Layer Nanoparticles

doi: 10.1021/acsnano.6c02817

Figure Lengend Snippet: HMW polyelectrolyte LbL films increase the half-life of IL-12 on cancer cell membranes via improved PLE presentation on LbL film. (a, b) Fluorescently labeled NPs were dosed to HM-1 cells in vitro . Four hours after dosing, cells were washed, and fresh cell culture media was added. After 0, 24, or 48 h after removing unbound NPs, cells were stained with anti-IL-12 monoclonal antibody, washed, and total IL-12 fluorescence associated with cells was evaluated on a fluorescence plate reader to determine an IL-12 fluorescence half-life. Shown are (a) the derived IL-12 half-life for the panel of PLR and PLE MW films (mean ± s.e.m.; significant differences not shown), and (b) the decay curves comparing the LWM (PLR 50 PLE 100 ) to the HMW (PLR 700 PLE 800 ) film (mean ± s.e.m.). (c) Fluorescently labeled NPs (cyan) were dosed to HM-1 cells in vitro . Two days (48 h) after dosing, cells were washed, fixed, and then stained with wheat germ agglutinin (cell membrane stain, green), Hoechst 33342 (nucleus stain, blue), and anti-IL-12 antibody (red). Cells were then visualized via confocal microscopy. Shown are representative confocal microscopy images of HM-1 cells treated with LMW or HMW LbL-NPs. (d) Snapshot of coarse-grained molecular dynamics simulation of LMW and HMW LbL films on a lipid bilayer membrane at 500 ns. (e) Train, loop, and tail populations computed at the residue level with fractions reported as per-chain means over adsorbed frames (50 to 500 ns), then averaged across included chains. Statistical comparisons performed using (b) pairwise comparison of curves with nonlinear regression and the extra-sum-of-squares F test. For each pair, the null hypothesis that the parameter k was identical across groups was tested using a threshold of p < 0.05 (**** p < 0.0001).

Article Snippet: HEK-Blue IL-12 (InvivoGen) cells were cultured and used for IL-12 bioactivity assessment according to the manufacturer’s instructions.

Techniques: Labeling, In Vitro, Cell Culture, Staining, Fluorescence, Derivative Assay, Membrane, Confocal, Microscopy, Residue, Comparison, Immunofluorescence

Journal: ACS Nano

Article Title: Polymer Molecular Weight Influences Cancer Cell Surface Retention and Cytokine Presentation by Layer-by-Layer Nanoparticles

doi: 10.1021/acsnano.6c02817

Figure Lengend Snippet: HMW polyelectrolyte LbL films increase cancerous tissue NP targeting and increase total IL-12 presentation. (a–h) B6C3F1 mice ( n = 4–5/group) inoculated with 10 6 HM-1-mCherrry-luc tumor cells on day 0 were administered fluorescently tagged NPs carrying 20 μg IL-12 (or an equivalent dose of free IL-12) on day 14. Four hours after dosing, animals were euthanized, ascites was collected and processed for flow cytometry, whereas tissues were analyzed ex vivo via IVIS. Shown are the median fluorescence intensity (MFI) of (a) NP signal in cancer cells (CD45 – mCherry + , mean ± s.d.) and (b) immune cells (CD45 + , mean ± s.d.), (c) the percentage of immune cells and cancer cells that were NP-positive (mean ± s.d.), (d) representative flow cytometry plot of NP and extracellular IL-12 fluorescence of tumor cells, (e) the percentage of CD45 + immune cells and HM-1 cancer cells that were IL-12-positive (mean ± s.d.), (f) the weight-normalized tissue NP fluorescence (mean ± s.e.m.), (g) Pearson’s correlation coefficient between weight-normalized tissue NP fluorescence and tumor BLI signal 4 h after dosing (mean ± s.e.m.), and (h) the significance of the correlation estimates (mean ± s.e.m.). Statistical comparisons performed using (a–c, e, g, h) one-way or (f) two-way ANOVA with Tukey’s multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: HEK-Blue IL-12 (InvivoGen) cells were cultured and used for IL-12 bioactivity assessment according to the manufacturer’s instructions.

Techniques: Tissue, Flow Cytometry, Ex Vivo, Fluorescence, Significance Assay, FACS

Journal: ACS Nano

Article Title: Polymer Molecular Weight Influences Cancer Cell Surface Retention and Cytokine Presentation by Layer-by-Layer Nanoparticles

doi: 10.1021/acsnano.6c02817

Figure Lengend Snippet: HMW polyelectrolytes increase LbL-NP treatment efficacy in a metastatic model of ovarian cancer. B6C3F1 mice ( n = 10/group) inoculated with 10 6 HM-1-luc tumor cells on day 0 were treated on days 7 and 14 with 20 μg of IL-12 as a free cytokine or conjugated to LbL-NPs. Shown are (a) the experimental timeline, (b) in vivo IVIS whole animal i.p. BLI readings (mean ± s.e.m.), and (c) overall survival. Statistical comparisons performed using (c) log-rank (Mantel–Cox) test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: HEK-Blue IL-12 (InvivoGen) cells were cultured and used for IL-12 bioactivity assessment according to the manufacturer’s instructions.

Techniques: In Vivo

Journal: ACS Nano

Article Title: Polymer Molecular Weight Influences Cancer Cell Surface Retention and Cytokine Presentation by Layer-by-Layer Nanoparticles

doi: 10.1021/acsnano.6c02817

Figure Lengend Snippet: Schematic of the proposed model illustrating how ionic strength during LbL assembly (top) and polymer molecular weight (bottom) independently govern LbL-NP film conformation and function. Increasing ionic strength produces thicker, more stable films that reduce polymer burst rearrangement and NP–NP interactions, but impairs IL-12 accessibility and cancer cell surface presentation. In contrast, increasing polymer MW stabilizes LbL films through outward extended chain conformations and increased adsorption avidity, delaying polymer rearrangement and minimizing NP–NP interactions while preserving IL-12 bioavailability, resulting in enhanced cancer cell binding and prolonged extracellular IL-12 presentation.

Article Snippet: HEK-Blue IL-12 (InvivoGen) cells were cultured and used for IL-12 bioactivity assessment according to the manufacturer’s instructions.

Techniques: Polymer, Molecular Weight, Adsorption, Preserving, Binding Assay, In Vivo