Journal: ACS nano

Article Title: Synthetic Charge-Invertible Polymer for Rapid and Complete Implantation of Layer-by-Layer Microneedle Drug Films for Enhanced Transdermal Vaccination

doi: 10.1021/acsnano.8b05373

Figure Lengend Snippet: (A) Film architecture schematics of [PAsp(EDDPA)/Cy5-OVA]40 LbL coated microneedle for mouse ear. (B) Confocal micrograph of Cy5-OVA on microneedle surface before and after 1 min application in mouse dermis. Scale bar 0.5 mm. (C) Image of mouse ear skin after 1 min microneedle (PDM/Polystyrene sulfonate/methylene blue)1 application showing the penetration pattern. Scale bar 2 mm. (D) Confocal micrograph of Cy5-OVA delivered onto mouse dermis after 15 s, 30 s, 60 s of microneedle insertion, respectively. Scale bar 1 mm. (E) Representative IVIS images showing Cy5 signal intensity on mouse ear at day 0, 1, 2, and 3 after MN or SC administration on left ear (right ear untreated). (F) Integrated radiant efficiency decay of Cy5-OVA on mouse ear in 3 days demonstrating in vivo sustained release of Cy5-OVA from PDM microneedle (MN) implanted LbL film, compared to subcutaneously (SC) injected same amount of Cy5-OVA (n = 6/group). Values are reported as mean ± s.e.m. ***p = 0.0004, as analyzed by one-way ANOVA. (G) Film architecture schematics of [Poly1/PolyI:C-TMR]40 LbL coated microneedle for human skin tissue. Flow cytometric analysis performed on cells isolated from human skin following 2 minute microneedle application for detection of film uptake, shown as (I) proportion, and (H) representative plots of antigen presenting cells (CD45+ CD14+ HLA-DR+) positive for PolyI:C-TMR LbL film; values are reported as mean ± s.e.m. **p=0.0017 unpaired t-test; numbers in plots indicate percent cells in outlined area.

Article Snippet: PolyI:C LbL coated microneedles for human skin were prepared using the StratoSequence VI from nanoStrata Inc. (USA) with spinning (100-120 rpm) 12 .

Techniques: In Vivo, Injection, Isolation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Interplay Between Liver Type 1 Innate Lymphoid Cells and NK Cells Drives the Development of Alcoholic Steatohepatitis

doi: 10.1016/j.jcmgh.2022.09.010

Figure Lengend Snippet: Injection of polyI:C protects mice from alcohol-induced steatohepatitis. ( A–D ) EtOH-fed mice were injected intraperitoneally with PBS or polyI:C every other day after ethanol feeding (5 mg/kg). Mice were killed, and liver tissue was obtained at 6 hours after binge. The numbers of liver ILC1 and NK cells ( A ) and ILC1/NK cells ratio ( B ) were shown (n = 3/group). ( C ) Hepatic triglyceride content was measured in whole liver homogenate (n = 4/group). ( D ) H&E staining of liver section was performed (scale bars, 100 μm). ( E ) Plasma levels of ALT and AST were detected (n = 5/group). ( F ) The mRNA level of IL1β and IL6 in liver tissue was detected (n = 4/group). Two-tailed unpaired Student t tests were performed in A and B . Representative images are shown (n = 4/group). Two-way analysis of variance was performed in A . Two-tailed unpaired Student t test was performed in B , C , E , and F . ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P <.0001.

Article Snippet: To expand NK cells in vivo, mice were injected intraperitoneally with 5 mg/kg of polyI:C every other day (APExBio, Houston, TX).

Techniques: Injection, Staining, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: RNase L Suppresses Androgen Receptor Signaling, Cell Migration and Matrix Metalloproteinase Activity in Prostate Cancer Cells

doi: 10.3390/ijms18030529

Figure Lengend Snippet: Hereditary Prostate Cancer 1-associated mutants of RNase L enhance AR transcriptional activity. ( A ) Endogenous RNase L-knockdown LNCaP cells were reconstituted with Flag-Wild-type (WT) or mutated RNase L as indicated and treated with vehicle (−R1881) or R1881 (1 nM) for 24 h. AR nuclear localization was analyzed under confocal microscope; and ( B ) quantitated by measuring fluorescence intensity using Image J software as described in B. White arrows point to lack of nuclear localization in WT and RNase L Y312A mutant expressing cells. AR transcriptional activity was monitored in LNCaP cells reconstituted with WT and RNase L mutants treated with vehicle or R1881 by ( C ) quantitative RT-PCR of mRNA levels of PSA , ETV1 and sGCα1 normalized to GAPDH mRNA levels. ( D ) PSA-luciferase promoter activity 18 h after R1881 treatment and normalized to β-galactosidase levels. Data shown are mean values ± SD of experiments performed in triplicate from three independent experiments. Student’s t- test was used to determine p -values. * p < 0.01, ** p < 0.001, # not significant, and compared to RNase L knockdown cells (shown with arrow on graphs) treated with R1881. ( E ) Cell lysates were analyzed for expression of Flag-RNase L (WT) and mutants as indicated using anti-Flag antibodies and normalized to β-actin levels. ( F ) HeLa cells were transfected with Flag-RNase L (WT) and RNase L mutants and enzyme activity was determined by monitoring rRNA cleavage (shown by arrows) as determined in RNA chips by transfecting cells with PolyI:C (2 μg/mL) and isolation of total RNA. Scale bar 10 µm, Magnification ×63.

Article Snippet: PolyI:C (2 μg/mL) was transfected into cells using Polyjet reagent (SignaGen Laboratories, Gaithersburg, MD, USA).

Techniques: Activity Assay, Microscopy, Fluorescence, Software, Mutagenesis, Expressing, Quantitative RT-PCR, Luciferase, Transfection, Isolation

Journal: Journal of Endotoxin Research

Article Title: Use of murine embryonic fibroblasts to define Toll-like receptor activation and specificity

doi: 10.1177/09680519040100060701

Figure Lengend Snippet: Fig. 1. Murine embryonic fibroblasts (MEFs) express mRNA for TLRs 1–9. MEFs were obtained from wild-type, TLR2 knockout and TLR4 knockout embryos. MEF mRNA was subjected to RT-PCR with primers specific for TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9. PCR products were separated by agarose gel electrophoresis and visualized with ethidium bromide. –RT, control lane without reverse transcriptase. Control lanes (–RT) were negative for all primers; + lane, GAPDH-positive control.

Article Snippet: TLR3 ligand – polyI:C was purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA) and used at 1 μM concentration.

Techniques: Knock-Out, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Control, Reverse Transcription, Positive Control

Journal: Journal of Endotoxin Research

Article Title: Use of murine embryonic fibroblasts to define Toll-like receptor activation and specificity

doi: 10.1177/09680519040100060701

Figure Lengend Snippet: Fig. 2. TLR2, TLR3 and TLR4 ligands induce cytokine secretion from MEFs. Wild-type MEFs were challenged with LPS (10 ng/ml), Pam3CSK4 (100 ng/ml), Pam2CSK4 (10 ng/ml), IL-1β (100 ng/ml), peptidoglycan (1 µg/ml), zymosan (10 µg/ml) or polyI:C (1 µM) for 18 h. IL-6 and MCP-1 secretion into culture supernatants was analyzed by ELISA. Data are representative of seven cell lines derived from individual embryos and from three cell lines derived from pools of 5–10 embryos.

Article Snippet: TLR3 ligand – polyI:C was purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA) and used at 1 μM concentration.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay