Rapid in vitro production of single-stranded DNA.

There is increasing demand for single-stranded DNA (ssDNA) of lengths >200 nucleotides (nt) in synthetic biology, biological imaging and bionanotechnology. Existing methods to produce high-purity long ssDNA face limitations in scalability, complexity of protocol steps and/or yield. We present a rapid, high-yielding and user-friendly method for in vitro production of high-purity ssDNA with lengths up to at least seven kilobases. Polymerase chain reaction (PCR) with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that ssDNA is recoverable in ∼40-50 min (time after PCR) with >70% yield with respect to the input PCR amplicon, or up to 70 pmol per 100 µl PCR reaction. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology directed repair in human cells, DNA-origami folding and fluorescent in-situ hybridization.

[Clinical Results of Tinted Aspherical Multifocal IOL with +2.5 Diopter Near Add Power SN6AD2 (SV25T0)].

PURPOSE: To evaluate the efficacy and safety of a newly developed diffractive multifocal intraocular lens (IOL) with +2.5 diopter near add power (SN6AD2, Alcon), whose theoretical distance for near focus is 50 cm. METHODS: This study included 128 eyes of 64 patients (23 men and 41 women, average age: 66.7 ± 7.2 years) following cataract removal at Tokyo Dental College Suidobashi Hospital and Hayashi Eye Hospital. Visual acuities (VAs) at distance, 1 m, 50 cm and 40 cm, and questionnaires were evaluated until 1 year postoperatively. RESULTS: At 1 year postoperatively, mean bilateral logMAR VAs were uncorrected -0.09 (5 m), 0.01 (1 m), 0.07 (50 cm), 0.18 (40 cm), distance corrected 0.01 (1 m), 0.08 (50 cm), 0.19 (40 cm), best corrected -0.17 (5 m), 0.00 (50 cm). From the patient's questionnaires, light or moderate glare was noticed in 17.5%, 4.8%, light or moderate halo was notice in 19.0%, 4.8% of the total patients. CONCLUSION: This IOL provides good distance to intermediate VAs and better near VA compared to monofocal IOL.

Intermittent weekly administration of human parathyroid hormone (1-34) improves bone-hydroxyapatite block bonding in ovariectomized rats.

Hydroxyapatite (HA) blocks have been widely used for the reconstruction of bone defects and as a bone substitute. Bone-implant bonding depends on both implant-related factors and patient variables. Intermittent human parathyroid hormone (h-PTH) has a strong anabolic effect on bone formation. The purpose of the present study is to evaluate whether intermittent h-PTH administration enhances bone-HA block bonding in normal versus ovariectomized (OVX) rats. Cancellous bone osteotomy and HA-block implantation were performed on the proximal left tibia in both OVX and sham-operated 7-month-old female Sprague-Dawley rats. Newly formed cancellous bone around the HA block and bone-HA block bonding were evaluated by bone histomorphometry at 8 weeks after the administration of h-PTH (100 µg/kg/week) or its vehicle. The administration of h-PTH significantly increased cancellous bone volume by stimulating bone formation in OVX rats (p < 0.01). Although bone-HA block bonding was significantly decreased in OVX rats compared to that of sham-operated rats (p < 0.01), h-PTH improved the bone-HA block bonding in OVX rats (p < 0.01). These results suggest that intermittent h-PTH treatment may improve bone-HA bonding in osteoporosis by restoring cancellous bone volume and enhancing cancellous bone formation around the HA block.

Polyvinyl alcohol-iodine induced corneal epithelial injury in vivo and its protection by topical rebamipide treatment.

Periocular povidone-iodine (PI) and polyvinyl alcohol-iodine (PAI) have had a major role in the prevention of endophthalmitis. The purpose of this study was to investigate the corneal epithelial toxicity of PAI in a rabbit eye model using corneal resistance (CR) measurement, which is a good indicator of cell barrier function. Rabbit eyes were administered PAI solution at 4-, 6-, 8-, or 16-fold dilution with physiological saline solution (saline) or saline alone (control), to the conjunctival sac with/without wash-out with saline. Corneal epithelial injury assessed by fluorescein staining and the CR ratio was measured at 10 minutes (min) to 96 hours (h) after the initial administration. Histological observation was performed in the eyes following the PAI or control administrations. At 120 min after administration of PAI solution, the CR ratio was decreased and superficial punctate keratopathy (SPK) was significantly increased in each of the PAI-administered groups compared to the control. Recovery of CR and SPK after administration of 6- or 8-fold dilution of PAI was significantly delayed in eyes that were not subsequently washed with saline compared with eyes that were. Pre- or post-instillation of 2% rebamipide ophthalmic suspension significantly reduced PAI induced-SPK and -decrease of CR ratio. The CR method was able to accurately and quantitatively evaluate fine corneal epithelial injury. It is suggested that eyes should be washed with saline solution after administration of PAI solution or the instillation of rebamipide to prevent or reduce corneal epithelial injury.

Optical mapping of the functional organization of the rat trigeminal nucleus: initial expression and spatiotemporal dynamics of sensory information transfer during embryogenesis.

We examined the functional organization of the rat trigeminal nuclear complex and its developmental dynamics using a multiple-site optical recording technique. Brainstem preparations were dissected from embryonic day 12 (E12)-E16 rat embryos, and stimulation was applied individually to the three branches of the trigeminal nerve (V1-V3). The action potential activity of presynaptic fibers was detected from E13, and the glutamate-mediated postsynaptic response was significantly observed from E15 on. At E14, the evoked signals usually consisted of only the action potential-related fast component. However, when extracellular Mg2+ was removed, a significant dl-2-amino-5-phosphonovaleric acid-sensitive slow component appeared. These results suggest that postsynaptic function mediated by NMDA receptors is latently generated as early as E14. The response area of the three branches of the trigeminal nerve showed some functional somatotopic organization, with the ophthalmic (V1) nerve area medially located and the mandibular (V3) nerve area laterally located. The center of the trigeminal nuclear complex in which the activity of neurons and synaptic function was greatest shifted caudally with development, suggesting that the functional architecture of the trigeminal nuclear complex is not fixed but changes dynamically during embryogenesis. By electron microscopy, we could not observe clear correlations between functional data and morphological information; when we surveyed E16 preparations, we could not identify typical synaptic structures between the 1,1'-dioctyldecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled trigeminal nerve terminals and the neurons in the trigeminal nuclear complex. This implies that postsynaptic function in the trigeminal nuclear complex is generated before the appearance of the morphological structure of conventional synapses.

Local administration of low-dose simvastatin-conjugated gelatin hydrogel for tendon-bone healing in anterior cruciate ligament reconstruction.

Anterior cruciate ligament (ACL) reconstruction with the hamstring tendon graft takes a long time, as the tendon graft needs to heal at the site of the bone-tendon integration in the created bone tunnels. Several reports have shown the therapeutic effects of simvastatin on bone formation with neovascularization. The aim of this study was to test the hypothesis that enhanced angiogenesis and osteogenesis by locally applied simvastatin promotes tendon-bone healing after ACL reconstruction. Rabbits received ACL reconstruction with hamstring tendon graft and were implanted with either simvastatin-conjugated gelatin hydrogel or gelatin hydrogel alone in their bone tunnels, and then bone regeneration and neovascularization at tendon-bone interface and biomechanical properties were assessed. Histological analysis at week 2 demonstrated that tendon-bone healing was significantly greater with angiogenesis and osteogenesis in the simvastatin-treated group than in the control group. Computed tomography at weeks 2 and 4 showed a significantly smaller tibial bone tunnel in the simvastatin-treated group. Biomechanical testing at week 2 demonstrated a significant increase in ultimate failure load in the simvastatin-treated group. This study suggested that local administration of low-dose simvastatin-conjugated gelatin hydrogel promotes the tendon-bone healing via its effect on both angiogenesis and osteogenesis at an early phase in a rabbit model, but does not affect biomechanical property in long-term after ACL reconstruction.

Corneal and retinal effects of ultraviolet-B exposure in a soft contact lens mouse model.

PURPOSE: To investigate the lipid and DNA oxidative stress as well as corneal and retinal effects after ultraviolet B (UV-B) exposure in mice, with or without silicon hydrogel soft contact lenses (SCL). METHODS: Twenty-eight C57BL6-strain male mice were divided into four groups: group I, control group with no SCL (SCL [-]) and no UV-B exposure (UV-B [-]); group II, senofilcon A SCL (senofilcon [+]) with UV-B exposure (UV-B [+]); group III, lotrafilcon A SCL (lotrafilcon [+]) with UV-B exposure (UV-B [+]); and group IV, no SCL (SCL [-]), but with UV-B exposure (UV-B [+]). All mice except group I received UV-B exposure for 5 days for a total dose of 2.73 J/cm(2). All mice underwent tear hexanoyl-lysine (HEL) and tear cytokine ELISA measurements, and fluorescein and rose bengal corneal staining before and after UV-B exposure. Corneal specimens underwent immunohistochemistry staining with CD45, HEL, 4-hydroxynonenal (4-HNE), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) antibodies and evaluation with electron microscopy. RESULTS: All mice without SCL but exposed to UV-B developed corneal edema, ulcers, or epithelial damage compared with mice with senofilcon A SCL and exposure to UV-B. Tear HEL and cytokine levels significantly increased in mice without SCL after UV-B exposure. Immunohistochemistry showed a significantly higher number of cells positively stained for CD45, 8-OHdG, HEL, and 4-HNE in the corneas of mice without SCLs compared with those with senofilcon A after UV-B exposure. CONCLUSION: Silicon hydrogel SCL showed corneal and retinal protective effects, owing to UV blocking properties, against oxidative stress-related membrane lipid and cellular DNA damage.