Comparison of the observable depth with fluorescent proteins ZsGreen1 (green) and tdTomato (red) in the liver treated with CUBIC.

<p>(A) Maximum intensity projections (X–Z plane). Scale bar: 100 μm. (B) Single plane images at the indicated depth (μm) from the liver surface. Scale bar: 50 μm. Acquisition conditions: lens, 20× dry; laser, 488 nm (output, 1%–12%; emission, 492–540 nm; master gain, 480–766) and 543 nm (output, 5%–100%; emission, 550–670 nm; master gain, 600–906).</p

Intracellular fate of plasmid DNA in transgene expression-positive cells of the liver treated with CUBIC after hydrodynamic injection.

<p>Three-dimensional maximum intensity projections and enlarged planes. Scale bar: 5 μm. Nuclei (blue), ZsGreen1 (cyan), Cy5-plasmid DNA (red), (A) TMR-dextran (green), and (B) TMR-plasmid DNA (green). Acquisition conditions: (A) lens, 63× oil-immersion; laser, 405 nm (output, 12.5%; emission, 425–460 nm; master gain, 781–862), 488 nm (output, 0.5%; emission, 503–533 nm; master gain, 580), 543 nm (output, 3.0%; emission, 548–627 nm; master gain, 1153) and 633 nm (output, 4.0%; emission, 639–748 nm; master gain, 1150); (B) lens, 63× oil-immersion; laser, 405 nm (output, 12.5%; emission, 414–480 nm; master gain, 752–815), 488 nm (output, 0.4%; emission, 525–531 nm; master gain, 525–531), 543 nm (output, 5.0%; emission, 585–647 nm; master gain, 1150), and 633 nm (output, 4.0%; emission, 639–690 nm; master gain, 1132).</p

Transmission color images of each tissue treated with CUBIC.

<p>Each lattice indicates 4×4 mm.</p

Observations of ZsGreen1 expression in various tissues treated with CUBIC.

<p>Three-dimensional maximum intensity projection. Acquisition conditions: lens, 20× dry (A, C, E, H, and J) or 40× oil-immersion (B, D, F, G, and I); laser, 488 nm; output, 5%; emission, 493–589 nm; master gain, (A) 951–1130, (B) 1081–1110, (C) 887–1152, (D) 732, (E) 823–977, (F) 732, (G) 1081–1110, (H) 1081–1110, (I) 1081–1110, and (J) 978–1146.</p

Comparison of tissue clearing methods.

<p>(A, E) PBS, (B, F) Clear^T2, (C, G) SeeDB, and (D, H) CUBIC. ZsGreen1 expression in the liver was detected by CLSM. (A–D) Depth coding. The color chart indicates depth on the Z-axis. (E–H) Maximum intensity projection (X–Z plane). Scale bar: 100 μm. Acquisition conditions: lens, 20× dry; laser, 488 nm; output, 5%; emission, 492–540 nm; master gain, (A, E) 432–1200, (B, F) 433–1129, (C, G) 405–1129, and (D, H) 492–1115.</p

Cell-Penetrating Peptides Using Cyclic α,α-Disubstituted α‑Amino Acids with Basic Functional Groups

In the delivery of cell-impermeable molecules, cell-penetrating peptides (CPPs) have been attracting increasing attention as intracellular delivery tools. In the present study, we designed four types of cyclic α,α-disubstituted α-amino acids (dAAs) with basic functional groups on their five-membered rings and different chiralities at the α-position and introduced them into arginine (Arg)-rich peptides. The evaluation of cell-penetrating abilities indicated that these peptides exhibited better cell permeabilities than an Arg nonapeptide. Furthermore, peptides containing dAAs delivered plasmid DNA (pDNA) better than a commercially available transfection reagent with a longer incubation time. These results demonstrate that the introduction of cyclic dAAs with basic functional groups into Arg-rich peptides is an effective strategy for the design of CPPs as a pDNA delivery tool

Cell-Penetrating Helical Peptides Having l‑Arginines and Five-Membered Ring α,α-Disubstituted α‑Amino Acids

Cell-penetrating peptides are powerful tools in the delivery of drugs, proteins, and nucleic acids into cells; therefore, focus has recently been placed on their development. In this study, we synthesized seven types of peptides possessing three l-arginines (l-Arg) and six l-leucines (l-Leu) and/or 1-aminocyclopentane-1-carboxylic acids (Ac₅c), and investigated their secondary structures and cell-penetrating abilities. The peptide composed of an equal number of l-Arg, l-Leu, and Ac₅c formed 3₁₀/α-helical structures in TFE solution and exhibited the highest cell-penetrating ability of all the peptides examined. Additional cellular uptake studies revealed that the incorporation of Ac₅c into peptides led to improved tolerability against serum. The results of the present study will help in the design of novel cell-penetrating peptides

Some Problems and Solutions in the Experimental Science of Technology: The Proper Use and Reporting of Statistics in Computational Intelligence, with an Experimental Design from Computational Ethnomusicology

Statistics is the meta-science that lends validity and credibility to The Scientific Method. However, as a complex and advanced Science in itself, Statistics is often misunderstood and misused by scientists, engineers, medical and legal professionals and others. In the area of Computational Intelligence (CI), there have been numerous misuses of statistical techniques leading to the publishing of insupportable results, which, in addition to being a problem in itself, has also contributed to a degree of rift between the Statistics/Statistical Learning community and the Machine Learning/Computational Intelligence community. This talk surveys a number of misuses of statistical inference in CI settings, including well-known and more rarely discussed examples. These are followed by an overview of concepts and techniques that are central to model evaluation. Finally, an experimental design is presented for a statistically valid comparison of multiple hypotheses for a particular real-world problem combining Information Theory, Neural Networks, Statistics, and Computational Ethnomusicology.https://pdxscholar.library.pdx.edu/systems_science_seminar_series/1060/thumbnail.jp

Efficient <i>in Vivo</i> Gene Transfer by Intraperitoneal Injection of Plasmid DNA and Calcium Carbonate Microflowers in Mice

Gene transfer to intraperitoneal organs is thought to be a promising approach to treat such conditions as peritoneal fibrosis and peritoneal dissemination of cancers. We previously discovered that simple instillation of naked plasmid DNA (pDNA) onto intraperitoneal organs such as the liver and stomach could effectively transfer foreign genes in mice. In this study, we developed a novel nonviral method to enhance transfection efficiency of naked pDNA to intraperitoneal organs using a calcium carbonate suspension containing pDNA. Using commercially available calcium carbonate, we successfully transfected pDNA to the stomach. Handling of commercially available calcium carbonate, however, was troublesome owing to rapid precipitation and caking. To obtain slowly settling particles of calcium carbonate, we tried to synthesize novel versions of such particles and succeeded in creating flower-shaped particles, named calcium carbonate microflowers. Sedimentation of calcium carbonate microflowers was sufficiently slow for <i>in vivo</i> experiments. Moreover, the transfection efficiency of the suspension of calcium carbonate microflowers to the stomach was more effective than that of commercially available calcium carbonate, especially at low concentrations. Intraperitoneal injection of the suspension of calcium carbonate microflowers containing pDNA greatly enhanced naked pDNA transfer to whole intraperitoneal organs in mice. Furthermore, lactate dehydrogenase activities in intraperitoneal fluid and plasma were not raised by the suspension of calcium carbonate microflowers