Epigenetic manipulation of gene expression: a toolkit for cell biologists

Cell biologists have been afforded extraordinary new opportunities for experimentation by the emergence of powerful technologies that allow the selective manipulation of gene expression. Currently, RNA interference is very much in the limelight; however, significant progress has also been made with two other approaches. Thus, antisense oligonucleotide technology is undergoing a resurgence as a result of improvements in the chemistry of these molecules, whereas designed transcription factors offer a powerful and increasingly convenient strategy for either up- or down-regulation of targeted genes. This mini-review will highlight some of the key features of these three approaches to gene regulation, as well as provide pragmatic guidance concerning their use in cell biological experimentation based on our direct experience with each of these technologies. The approaches discussed here are being intensely pursued in terms of possible therapeutic applications. However, we will restrict our comments primarily to the cell culture situation, only briefly alluding to fundamental differences between utilization in animals versus cells

Ultrathin, polarization-independent, and focus-tunable liquid crystal diffractive lens for augmented reality

Despite the recent advances in augmented reality (AR), which has shown the potential to significantly impact on our daily lives by offering a new way to manipulate and interact with virtual information, minimizing visual discomfort due to the vergence-accommodation conflict remains a challenge. Emerging AR technologies often exploit focus-tunable optics to address this problem. Although they demonstrated improved depth perception by enabling proper focus cues, a bulky form factor of focus-tunable optics prevents their use in the form of a pair of eyeglasses. Herein, we describe an ultrathin, focus-tunable liquid crystal (LC) diffractive lens with a large aperture, a low weight, and a low operating voltage. In addition, we show that the polarization dependence of the lens, which is an inherent optical property of LC lenses, can be eliminated using birefringent thin films as substrates and by aligning the optical axes of the birefringent substrates and LC at a specific angle. The polarization independence eliminates the need for a polarizer, thus further reducing the form factor of the optical system. Next, we demonstrate a prototype of AR glasses with addressable focal planes using the ultrathin lens. The prototype AR glasses can adjust the accommodation distance of the virtual image, mitigating the vergence-accommodation conflict without substantially compromising the form factor or image quality. This research on ultrathin lens technology shows promising potential for developing compact optical displays in various applications.Comment: 23 pages, 9 figure

Cellular Delivery and Biological Activity of Antisense Oligonucleotides Conjugated to a Targeted Protein Carrier

Targeted delivery can potentially improve the pharmacological effects of antisense and siRNA oligonucleotides. Here we describe a novel bioconjugation approach to the delivery of splice-shifting antisense oligonucleotides (SSOs). The SSOs are linked to albumin via reversible S-S bonds. The albumin is also conjugated with polyethylene glycol (PEG) chains that terminate in an RGD ligand that selectively binds the αvβ3 integrin. As a test system we utilized human melanoma cells that express the αvβ3 integrin and that also contain a luciferase reporter gene that can be induced by delivery of SSOs to the cell nucleus. The RGD-PEG-SSO-albumin conjugates were endocytosed by the cells in an RGD-dependent manner; using confocal fluorescence microscopy evidence was obtained that the SSOs accumulate in the nucleus. The conjugates were able to robustly induce luciferase expression at concentrations in the 25−200nM range. At these levels little short-term or long-term toxicity was observed. Thus the RGD-PEG-Albumin conjugates may provide an effective tool for targeted delivery of oligonucleotides to certain cells and tissues

Stenting of the Left Main Coronary Artery in a Patient With Takayasu's Arteritis

Management of Takayasu's arteritis of the left main coronary artery (LMCA) is difficult because of the possibility of restenosis. Clinically significant stenotic lesions must be considered anatomical correlation. Many studies have reported that the management of stenotic lesions of the LMCA with endoluminal stenting and balloon angioplasty and de-novo stenting is safe and effective for patients with Takayasu's arteritis. We report the case of a patient with Takayasu's arteritis of the LMCA. The patient had undergone two consecutive percutaneous coronary interventions because of recurrent restenosis of in-stent lesions, and eventually underwent coronary artery bypass graft (CABG) surgery for myocardial infarction in the same lesion. We suggested treatment with CABG because the pathophysiology of Takayasu's arteritis is different from that of atherosclerotic stenosis

Osseointegration of Implants Surface-Treated with Various Diameters of TiO 2

The aim of this study was to evaluate the osseointegration of implants which were surface-treated with various diameters of TiO2 nanotubes (30 nm, 70 nm, and 100 nm) in rabbit. Resorbable blast media (RBM) surfaced implants (Osstem, Busan, Korea) 3.5 mm in diameter and 8.5 mm in length were designated as the control group and the implants surface-treated with various diameters of nanotubes (30 nm, 70 nm, and 100 nm) with the same shapes were designated as the experimental groups. The implants were maintained unloaded for 4 and 12 weeks. After this period, the animals were sacrificed and micro-CT analysis, histomorphometric analysis (bone to implant contact (BIC), bone volume (BV)), and removal torque test were performed. Micro-CT analysis, histomorphometric analysis, and removal torque test results all showed the similar pattern, showing that 70 nm experimental group had the highest value at 4 weeks while 30 nm experimental group had the highest value at 12 weeks. Therefore, on the basis of the results above, it can be concluded that 30 nm and 70 nm TiO2 nanotubes may have positive effects on osteogenesis and osseointegration depending on the healing time

Validation of the finger counting method using the Monte Carlo simulation

Purpose The dose of drug and the size of instrument are determined based on children’s weight. We aimed to validate the finger counting method (FCM) for weight estimation in Korean children using the Monte Carlo simulation. Methods We estimated the weight of Korean children aged 1 to 9 years by the FCM. These measurements were compared with the weight extracted by the Monte Carlo simulation applied to the “2007 Korean Children and Adolescents Growth Standard.” Pearson correlation coefficients (r) were measured to assess the correlation between the weight extracted by the simulation and that estimated by FCM. Bland-Altman analyses were performed to assess the agreement between the weight extracted by the simulation and that estimated by FCM and 2 other well-known pediatric weight estimation formulas (the Advanced Pediatric Life Support and Luscombe formulas). Results Data regarding 9,000 children’s weight selected by age and gender was randomly extracted using the simulation. We found a positive correlation between the weight estimated by the FCM and the weight extracted (in boys, r = 0.896, P < 0.001; in girls, r = 0.899, P < 0.001). The FCM tended to underestimate weight in the children aged 7 years or old. Conclusion This article suggests the usefulness of FCM in weight estimation, particularly in children younger than 7 years. With appreciation of the limitation in older children, the FCM could be applied to emergency practice

Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis

We describe the synthesis and characterization of a 5′ conjugate between a 2′-O-Me phosphorothioate antisense oligonucleotide and a bivalent RGD (arginine–glycine–aspartic acid) peptide that is a high-affinity ligand for the αvβ3 integrin. We used αvβ3-positive melanoma cells transfected with a reporter comprised of the firefly luciferase gene interrupted by an abnormally spliced intron. Intranuclear delivery of a specific antisense oligonucleotide (termed 623) corrects splicing and allows luciferase expression in these cells. The RGD–623 conjugate or a cationic lipid-623 complex produced significant increases in luciferase expression, while ‘free’ 623 did not. However, the kinetics of luciferase expression was distinct; the RGD–623 conjugate produced a gradual increase followed by a gradual decline, while the cationic lipid-623 complex caused a rapid increase followed by a monotonic decline. The subcellular distribution of the oligonucleotide delivered using cationic lipids included both cytoplasmic vesicles and the nucleus, while the RGD–623 conjugate was primarily found in cytoplasmic vesicles that partially co-localized with a marker for caveolae. Both the cellular uptake and the biological effect of the RGD–623 conjugate were blocked by excess RGD peptide. These observations suggest that the bivalent RGD peptide–oligonucleotide conjugate enters cells via a process of receptor-mediated endocytosis mediated by the αvβ3 integrin