Global landscape of replicative DNA polymerase usage in the human genome.

The division of labour among DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequencing (Pu-seq) in HCT116 cells and mapped Polε and Polα usage genome wide. The polymerase usage profiles show Polε synthesises the leading strand and Polα contributes mainly to lagging strand synthesis. Combining the Polε and Polα profiles, we accurately predict the genome-wide pattern of fork directionality plus zones of replication initiation and termination. We confirm that transcriptional activity contributes to the pattern of initiation and termination and, by separately analysing the effect of transcription on co-directional and converging forks, demonstrate that coupled DNA synthesis of leading and lagging strands is compromised by transcription in both co-directional and convergent forks. Polymerase uncoupling is particularly evident in the vicinity of large genes, including the two most unstable common fragile sites, FRA3B and FRA3D, thus linking transcription-induced polymerase uncoupling to chromosomal instability. Together, our result demonstrated that Pu-seq in human cells provides a powerful and straightforward methodology to explore DNA polymerase usage and replication fork dynamics.

Switching of Photocatalytic Tyrosine/Histidine Labeling and Application to Photocatalytic Proximity Labeling.

Weak and transient protein interactions are involved in dynamic biological responses and are an important research subject; however, methods to elucidate such interactions are lacking. Proximity labeling is a promising technique for labeling transient ligand-binding proteins and protein-protein interaction partners of analytes via an irreversible covalent bond. Expanding chemical tools for proximity labeling is required to analyze the interactome. We developed several photocatalytic proximity-labeling reactions mediated by two different mechanisms. We found that numerous dye molecules can function as catalysts for protein labeling. We also identified catalysts that selectively modify tyrosine and histidine residues and evaluated their mechanisms. Model experiments using HaloTag were performed to demonstrate photocatalytic proximity labeling. We found that both ATTO465, which catalyzes labeling by a single electron transfer, and BODIPY, which catalyzes labeling by singlet oxygen, catalyze proximity labeling in cells.

A novel gradient and multilayered sheet with a silk fibroin/polyvinyl alcohol core-shell structure for bioabsorbable arterial grafts.

Bioabsorbable arterial grafts can potentially improve patency and neovessel formation; however, their application in clinical settings has not been realized. In this study, we developed bioabsorbable gradient sheets based on silk fibroin (SF) and polyvinyl alcohol (PVA) with a core-shell nanofibrous structure. This gradient sheet was expected to promote vascular remodeling while we maintained its physical properties and a gradual degrading process from the luminal surface. ESP was conducted at various flow rates for SF and PVA to achieve the multilayer gradient structure. Furthermore, the elasticity of the gradient sheet could be increased by increasing the PVA flow rate; however, this reduced the tensile strength of the core-shell fibers. Notably, the physical properties of the gradient sheet did not degrade even after 7 days of immersion in a phosphate buffer saline solution, which indicates that the structure could maintain its structural integrity while resisting arterial pressure. In vitro experiments revealed that the number of endothelial cells attached to the SF/PVA sheet was notably higher than that on the cell-culture dish. The gradient sheets were implanted in rat abdominal aortas and explanted after 14 days to confirm acute-phase patency and vascular remodeling. The gradient sheets constructed with SF composed of polyurethane and PVA improved the ease of handling of the material, and these sheets resulted in a favorable vascular remodeling outcome. Our results strongly suggest that the SF/PVA-based gradient sheets described in this study can serve as a novel design for bioabsorbable arterial grafts upon further modifications.

Pharmacokinetic and Safety Evaluation of a Transscleral Sustained Unoprostone Release Device in Monkey Eyes.

Purpose: We evaluate the ocular tissue distribution and retinal toxicity of unoprostone (UNO) during 12 months, after transscleral sustained-UNO administration using a drug delivery device in monkey eyes. Methods: The device consisted of a reservoir, controlled-release cover, and a drug formulation of photopolymerized polyethylene glycol dimethacrylate. Six mg UNO was loaded into the device (length, 17 mm; width, 4.4 mm; height, 1 mm). The concentrations of M1, a primary metabolite of UNO, in the retina, choroid, vitreous, lens, aqueous humor, iris, ciliary body, and plasma were determined by liquid chromatography-tandem mass spectrometry at 3, 6, and 12 months after implantation. Retinal toxicity was evaluated by electroretinography (ERG), optical coherence tomography (OCT), and IOP at preimplantation, and at 6, 9, and 12 months after implantation. Focal ERGs were performed at 9 and 12 months after implantation. Results: M1 was detected in the choroid and retina with maximum peaks of 243.2 and 8.41 ng/g at 6 months, respectively. M1 in the ciliary body and iris was detected with maximum peaks of 7.66 and 10.4 ng/g at 6 and 12 months, respectively. Less than 1 ng/mL or ng/g of M1 was detected in the aqueous humor, vitreous, and lens. No changes were observed in retinal function as assessed by ERG, IOP, or macula thickness and retinal histology by OCT examinations during the 12-month period. No differences in focal ERG amplitudes, especially in the macula, were observed. Conclusions: The device provided intraocular sustained delivery of UNO for 12 months without producing severe retinal toxicity.

Long-Term Protection of Genetically Ablated Rabbit Retinal Degeneration by Sustained Transscleral Unoprostone Delivery.

Purpose: To evaluate the long-term protective effects of transscleral unoprostone (UNO) against retinal degeneration in transgenic (Tg) rabbits (Pro347Leu rhodopsin mutation). Methods: The UNO release devices (URDs) were implanted into the sclerae of Tg rabbits and ERG, optical coherence tomography (OCT), and ophthalmic examinations were conducted for 40 weeks. Unoprostone metabolites in retina, choroid/RPE, aqueous humor, and plasma from wild-type (Wt) rabbits were measured using liquid chromatography-tandem mass spectrometry. In situ hybridization and immunohistochemistry evaluated the retinal distribution of big potassium (BK) channels, and RT-PCR evaluated the expressions of BK channels and m-opsin at 1 week after URD treatment. Results: The URD released UNO at a rate of 10.2 ±1.0 µg/d, and the release rate and amount of UNO decreased during 32 weeks. Higher ERG amplitudes were observed in the URD-treated Tg rabbits compared with the placebo-URD, or nontreated controls. At 24 weeks after implantation into the URD-treated Tg rabbits, OCT images showed preservation of retinal thickness, and histologic examinations (44 weeks) showed greater thickness of outer nuclear layers. Unoprostone was detected in the retina, choroid, and plasma of Wt rabbits. Retina/plasma ratio of UNO levels were 38.0 vs. 0.68 ng UNO*hour/mL in the URD-treated group versus control (topical UNO), respectively. Big potassium channels were observed in cone, cone ON-bipolar, and rod bipolar cells. Reverse-transcriptase PCR demonstrated BK channels and m-opsins increased in URD-treated eyes. Conclusions: In Tg rabbits, URD use slowed the decline of retinal function for more than 32 weeks, and therefore provides a promising tool for long-term treatment of RP.

Age-dependent differences in recovered visual responses in Royal College of Surgeons rats transduced with the Channelrhodopsin-2 gene.

The objective of this study is to investigate age-related differences in recovered visual function in Royal College of Surgeons (RCS) rats transduced with the Channelrhodopsin-2 (ChR2) gene. An adeno-associated virus vector that contained ChR2 was injected intravitreously into young or aged RCS rats. After 4 months, visual evoked potentials were recorded. To estimate the transduction efficiencies, ChR2V-expressing cells and retrograde labeled retinal ganglion cells (RGCs) were counted. After photoreceptor degradation, immunohistochemistry was used to detect glial fibrillary acidic protein (GFAP) in the retinas. The amplitudes and latencies from young RCS rats were higher and shorter, respectively, than those from aged RCS rats. ChR2V was expressed in the RGCs of both groups of rats; there was no significant difference in the transduction efficiency of either group. However, the number of RGCs in aged RCS rats was significantly less than that in young RCS rats. In addition, strong GFAP immunoreactivity was observed after photoreceptor degeneration, whereas it was weaker in ChR2V-expressing RGCs. ChR2 transduction produced photosensitive RGCs in both young and aged rats. However, the degree of recovery depended on the age at the time of transduction.