Journal: JACC: Basic to Translational Science

Article Title: Efficacy and Safety of Dual Paclitaxel and Sirolimus Nanoparticle-Coated Balloon

doi: 10.1016/j.jacbts.2024.02.002

Figure Lengend Snippet: In Vitro Cell Culture Studies Human vascular smooth muscle cells were treated at IC 50 (ie, the dose required to achieve 50% inhibition of cell proliferation with respect to untreated cells) drug doses in nanoparticles (NPs) for 3 days. Different treatments were control NPs, sirolimus (SRL) NPs (3,100 ng/mL), paclitaxel (PTX) NPs (45 ng/mL), and dual active pharmaceutical ingredient (API) NPs (225 ng/mL). (A) Microscopic observation and quantitative analysis of apoptotic/dead cells following treatment with control NPs, SRL NPs, PTX NPs, and dual API NPs. Red arrowheads indicate the apoptotic/dead cells. Bar = 10 μm. The data show significantly higher apoptotic/dead cells in PTX NPs than in other treatment groups. (B) Quantification of proliferating cells. The BrdU-positive cells following different drug treatments were imaged and quantified. Red arrowheads in the images show proliferating cells. Bar = 20 μm. The quantitative analysis shows a significantly lower number of bromodeoxyuridine (BrdU)-positive cells in dual API NPs compared to control or SRL NPs. (C) Flow cytometric analysis. Cells following different treatments were analyzed for different phases of cell cycle. The results show that dual API NPs cause the inhibition of cell proliferation by cell cycle arrest (G0/G1). Different phases of the cell cycle. G0/G1 phase = cells exit G1 and enter a resting state/cell cycle arrest phase called G0 (SRL mechanism); G2/M phase = gap 2 (G2); M phase= mitosis (PTX mechanism); S phase = DNA synthesis for cell replication or proliferative phase. Values are mean ± SEM. Differences in the groups were calculated using the Student’s t -test. ∗∗∗ P = 0.001.

Article Snippet: Cells were cultured in a smooth muscle cell growth medium (Cat# 311-500, Cell Applications Inc) at 37 °C in a humidified 5% CO 2 atmosphere.

Techniques: In Vitro, Cell Culture, Inhibition, Control, DNA Synthesis

Journal: JACC: Basic to Translational Science

Article Title: Efficacy and Safety of Dual Paclitaxel and Sirolimus Nanoparticle-Coated Balloon

doi: 10.1016/j.jacbts.2024.02.002

Figure Lengend Snippet: Representative Histology of Rabbit Iliac Arteries 5 Days After the Treatment With Dual API DCB or PTX DCB Histology sections from iliac arteries treated with (A) the dual API DCB or (B) the PTX DCB. (Upper) Low-power images of (A-i and B-i) Movat pentachrome-stained and (A-ii and B-ii) BrdU-stained images. (Middle) High-power images of (A-iii and B-iii) hematoxylin and eosin (H&E) and (A-iv and B-iv) BrdU staining from blue rectangular boxes seen in upper images. (Bottom) High-power images of (A-v and B-v) H&E and (A-vi and V-vi) BrdU staining from red rectangular boxes in upper images. There is (A-iii and A-v) smooth muscle cell (SMC) loss with (A-iv) BrdU-positive nuclei (arrows) in the media of the section treated with the dual API DCB, whereas the section treated with the PTX DCB showed (B-iii) severe loss of SMC without (B-iv) BrdU-positive nuclei in media. (A-iii to A-vi) There were no nuclei on the luminal surface in the section treated with the dual API DCB, whereas (B-iv and B-vi) BrdU-positive nuclei were observed in the neointimal area of the section treated with the PTX DCB (arrows). Abbreviations as in Figure 1 .

Article Snippet: Cells were cultured in a smooth muscle cell growth medium (Cat# 311-500, Cell Applications Inc) at 37 °C in a humidified 5% CO 2 atmosphere.

Techniques: Staining, BrdU Staining

Journal: Virulence

Article Title: Synthetic Scrapie Infectivity: Interaction between Recombinant PrP and Scrapie Brain-Derived RNA

doi: 10.4161/21505594.2014.989795

Figure Lengend Snippet: Bioassay results for the phenol/chloroform group: first and second experiments

Article Snippet: None of the animals in any of the other recipient groups challenged with either SAF-derived control samples (inocula 2, 6, 7 and 11) or negative controls (inocula 12 and 13) developed scrapie symptoms during a long observation period of 545 dpi ( ). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Inocula a Description Scrapie after IC Death from other causes Residual infectivity WB PrP sc Incubation periods b 1 BioRad purified scrapie-associated fibrils 4/4 0/4 N/A + 85, 85, 85, 85 Experiment 1: SAF treated with PK and phenol/chloroform (duration: 545 days) 2 Phenol/chloroform aqueous phase (Ph/Ch) 0/5 1/5 φ No φ + 413 φ 6 Ph/Ch + β-recPrP oligomers 0/7 2/7 No − 300*, 424* 7 Ph/Ch + α-recPrP 0/5 1/5 No − 310* 8 Ph/Ch + DNase I + β-recPrP oligomers 2/5 1/5 No + 151, 246, 452* 11 Ph/Ch + β-recPrP fibrils 0/7 3/7 No - 424*, 508*, 515* 12 α - recPrP monomers 0/4 c 0/4 No 545 c 13 β- recPrP oligomers 0/2 1/2 No 410* Experiment 2: SAF treated with PK and phenol/chloroform (duration: 480 days) A Ph/Ch II 0/7 0/7 No B Ph/Ch II + DNase I 0/17 1/17 No − 279* C Ph/Ch II + DNase I + α- recPrP monomers 0/5 0/5 No D Ph/Ch II +DNase I + β-recPrP oligomers 3/19 0/19 No + 148, 178, 207 Open in a separate window Note: a, Inoculations were performed while avoiding cross contamination; for example, infectious inoculum 8 was inoculated following the 2 negative inocula 6 and 7. b, Period of time between injection and death/sacrifice upon the appearance of severe clinical signs in the animal (ataxia, rachitis, tremor). c, All animals that did not develop symptoms were sacrificed 545 days post inoculation.

Techniques: Infection, Incubation, Purification

Journal: Virulence

Article Title: Synthetic Scrapie Infectivity: Interaction between Recombinant PrP and Scrapie Brain-Derived RNA

doi: 10.4161/21505594.2014.989795

Figure Lengend Snippet: Nucleic acid analysis and formation of recPrP nanostructures. (A) Electropherogram of inoculum 8 (DNase I-digested inoculum 2) analyzed using a small RNA assay chip, showing 2 distinct populations of RNA with average lengths of approximately 27 and 55 nucleotides. Approximate quantities of oligonucleotides measured in each peak are 0.5 ng for the marker, 0.2 ng for the first peak (∼27 nt) and 0.07 ng for the second peak (∼55 nt). (B) Electropherogram of DNase I-digested inoculum 2 analyzed using a large RNA integrity assay chip, showing the absence of large cellular RNA and degradation products. (C) Inoculum 2 analyzed using a small RNA assay chip, showing the presence of cellular DNA (sizes ≤ 300 nucleotides). Approximately 37 ng of DNA is observed for 0.16 mg brain equivalent loaded. (D) Electron micrograph of inoculum 8 after negative staining, indicating the presence of ovoid and spherical aggregates of PrP with diameters of 7-40 nm. (E) Electron micrograph of control inoculum 7 (Ph/Ch + α-recPrP monomers), showing the absence of PrP aggregates (scale bar, 100 nm). For (A-C) 1 μl of sample was loaded onto the chip, which is approximately equal to 8 ng equivalent PrPSc and approximately 0.16 mg brain equivalent.

Article Snippet: None of the animals in any of the other recipient groups challenged with either SAF-derived control samples (inocula 2, 6, 7 and 11) or negative controls (inocula 12 and 13) developed scrapie symptoms during a long observation period of 545 dpi ( ). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Inocula a Description Scrapie after IC Death from other causes Residual infectivity WB PrP sc Incubation periods b 1 BioRad purified scrapie-associated fibrils 4/4 0/4 N/A + 85, 85, 85, 85 Experiment 1: SAF treated with PK and phenol/chloroform (duration: 545 days) 2 Phenol/chloroform aqueous phase (Ph/Ch) 0/5 1/5 φ No φ + 413 φ 6 Ph/Ch + β-recPrP oligomers 0/7 2/7 No − 300*, 424* 7 Ph/Ch + α-recPrP 0/5 1/5 No − 310* 8 Ph/Ch + DNase I + β-recPrP oligomers 2/5 1/5 No + 151, 246, 452* 11 Ph/Ch + β-recPrP fibrils 0/7 3/7 No - 424*, 508*, 515* 12 α - recPrP monomers 0/4 c 0/4 No 545 c 13 β- recPrP oligomers 0/2 1/2 No 410* Experiment 2: SAF treated with PK and phenol/chloroform (duration: 480 days) A Ph/Ch II 0/7 0/7 No B Ph/Ch II + DNase I 0/17 1/17 No − 279* C Ph/Ch II + DNase I + α- recPrP monomers 0/5 0/5 No D Ph/Ch II +DNase I + β-recPrP oligomers 3/19 0/19 No + 148, 178, 207 Open in a separate window Note: a, Inoculations were performed while avoiding cross contamination; for example, infectious inoculum 8 was inoculated following the 2 negative inocula 6 and 7. b, Period of time between injection and death/sacrifice upon the appearance of severe clinical signs in the animal (ataxia, rachitis, tremor). c, All animals that did not develop symptoms were sacrificed 545 days post inoculation.

Techniques: Marker, Integrity Assay, Negative Staining

Journal: Virulence

Article Title: Synthetic Scrapie Infectivity: Interaction between Recombinant PrP and Scrapie Brain-Derived RNA

doi: 10.4161/21505594.2014.989795

Figure Lengend Snippet: Testing for PrPSc seeding activity in phenol/chloroform extracts and bioassay samples (A) Testing phenol/chloroform extracts (Ph/Ch) from a crude preparation of 263K scrapie-associated fibrils that had been purified using a previously published protocol31 for residual PrPSc seeding activity using serial PMCA (the third and fourth PMCA rounds are shown). Lanes 1: PK digested 263K scrapie hamster brain homogenate containing 5 × 10−7 g of 263K hamster brain tissue. Lanes 2–8: Reference PMCA samples seeded with 1 × 10−12 (lanes 2 and 3), 1 × 10−11 (lanes 4 and 5), 1 × 10−10 (lanes 6 and 7) or 1 × 10−9 g 263K hamster brain tissue (lane 8). Lanes 9 and 10: Phenol/chloroform extracts isolated from uninfected hamster brain. Lanes 11 and 12: Ph/Ch extracts isolated from 263K scrapie hamster brain (corresponding to ∼500 ng 'equivalent PrPSc' per lane, i.e., to an approximately 2.5-fold amount of the intracerebral inoculum administered to hamsters, which was 200 ng 'equivalent PrPSc'). Lanes 13 and 14: Ph/Ch extract isolated from 263K scrapie hamster brain spiked with 1 × 10−11 g of 263K scrapie brain tissue following phenol/chloroform extraction. (B) Testing various bioassay inocula for the amount of residual or newly produced PrP seeding activity using serial PMCA. Lanes 1, 8, 15 and 19: PK-digested 263K scrapie hamster brain homogenate containing 5 × 10−7 g of 263K hamster brain tissue. Lanes 2–7: Reference PMCA samples seeded with 1 × 10−12 (lanes 2 and 3), 1 × 10−11 (lanes 4 and 5), 1 × 10−10 (lane 6) or 1 × 10−9 g 263K hamster brain tissue (lane 7). Lanes 9–14: Negative controls without seeding material (i.e., PMCA with normal hamster brain homogenate only). Lane 16: PMCA seeded with 10 μl of inoculum 6 (Ph/Ch + β-rec PrP oligomers). Lane 17: PMCA seeded with 6 μl of inoculum 7 (Ph/Ch + α-rec PrP monomers). Lane 18: PMCA seeded with 6 μl of inoculum 8 (Ph/Ch + DNase I + β-rec PrP oligomers). Lanes 20–22: Same samples as in lanes 16–18, spiked with 1 × 10−10 g of 263K scrapie brain tissue.

Article Snippet: None of the animals in any of the other recipient groups challenged with either SAF-derived control samples (inocula 2, 6, 7 and 11) or negative controls (inocula 12 and 13) developed scrapie symptoms during a long observation period of 545 dpi ( ). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Inocula a Description Scrapie after IC Death from other causes Residual infectivity WB PrP sc Incubation periods b 1 BioRad purified scrapie-associated fibrils 4/4 0/4 N/A + 85, 85, 85, 85 Experiment 1: SAF treated with PK and phenol/chloroform (duration: 545 days) 2 Phenol/chloroform aqueous phase (Ph/Ch) 0/5 1/5 φ No φ + 413 φ 6 Ph/Ch + β-recPrP oligomers 0/7 2/7 No − 300*, 424* 7 Ph/Ch + α-recPrP 0/5 1/5 No − 310* 8 Ph/Ch + DNase I + β-recPrP oligomers 2/5 1/5 No + 151, 246, 452* 11 Ph/Ch + β-recPrP fibrils 0/7 3/7 No - 424*, 508*, 515* 12 α - recPrP monomers 0/4 c 0/4 No 545 c 13 β- recPrP oligomers 0/2 1/2 No 410* Experiment 2: SAF treated with PK and phenol/chloroform (duration: 480 days) A Ph/Ch II 0/7 0/7 No B Ph/Ch II + DNase I 0/17 1/17 No − 279* C Ph/Ch II + DNase I + α- recPrP monomers 0/5 0/5 No D Ph/Ch II +DNase I + β-recPrP oligomers 3/19 0/19 No + 148, 178, 207 Open in a separate window Note: a, Inoculations were performed while avoiding cross contamination; for example, infectious inoculum 8 was inoculated following the 2 negative inocula 6 and 7. b, Period of time between injection and death/sacrifice upon the appearance of severe clinical signs in the animal (ataxia, rachitis, tremor). c, All animals that did not develop symptoms were sacrificed 545 days post inoculation.

Techniques: Activity Assay, Purification, Isolation, Produced