Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation.

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases

Luminal-contact-inhibition of epithelial basal stem cell multipotency in prostate organogenesis and homeostasis.

Prostate epithelial basal cells are highly plastic in their luminal differentiation capability. Basal stem cells actively produce luminal cells during organogenesis, but become restricted in the adult prostate unless receiving oncogenic or inflammatory stimuli. Given that the number of luminal cells increases relative to basal cells through development and that equilibrium is reached in the adulthood, we hypothesize that a negative-feedback mechanism exists to inhibit basal-to-luminal differentiation. We provide evidence supporting this hypothesis by comparing murine prostatic growth in a tissue reconstitution assay with cell recombinants of different basal-to-luminal ratios. Additionally, in organoid culture, hybrid organoids derived from adjacent basal and luminal cells showed reduced basal stem cell activities, suggesting contact inhibition. Importantly, removal of adult luminal cells in vivo via either an inducible Cre/loxP-Dre/rox dual-lineage-tracing system or orthotopic trypsin injection led to robust reactivation of basal stem cell activities, which acts independent of androgen. These data illustrate the prostate organ as a distinctive paradigm where cell contact from differentiated daughter cells restricts adult stem cell multipotency to maintain the steady-state epithelial architecture

Documentation of Intraretinal Retinal Pigment Epithelium Migration via High-Speed Ultrahigh-Resolution Optical Coherence Tomography

Purpose To describe the features of intraretinal retinal pigment epithelium (RPE) migration documented on a prototype spectral-domain, high-speed, ultrahigh-resolution optical coherence tomography (OCT) device in a group of patients with early to intermediate dry age-related macular degeneration (AMD) and to correlate intraretinal RPE migration on OCT to RPE pigment clumping on fundus photographs. Design Retrospective, noncomparative, noninterventional case series. Participants Fifty-five eyes of 44 patients seen at the New England Eye Center between December 2007 and June 2008 with early to intermediate dry AMD. Methods Three-dimensional OCT scan sets from all patients were analyzed for the presence of intraretinal RPE migration, defined as small discreet hyperreflective and highly backscattering lesions within the neurosensory retina. Fundus photographs also were analyzed to determine the presence of RPE pigment clumping, defined as black, often spiculated, areas of pigment clumping within the macula. The en face OCT images were correlated with fundus photographs to demonstrate correspondence of intraretinal RPE migration on OCT and RPE clumping on fundus photography. Main Outcome Measures Drusen, dry AMD, intraretinal RPE migration, and RPE pigment clumping. Results On OCT scans, 54.5% of eyes (61.4% of patients) demonstrated intraretinal RPE migration. Of the fundus photographs, 56.4% demonstrated RPE pigment clumping. All eyes with intraretinal RPE migration on OCT had corresponding RPE pigment clumping on fundus photographs. The RPE pigment migrated most frequently into the outer nuclear layer (66.7% of eyes) and less frequently into more anterior retinal layers. Intraretinal RPE migration mainly occurred above areas of drusen (73.3% of eyes). Conclusions The appearance of intraretinal RPE migration on OCT is a common occurrence in early to intermediate dry AMD, occurring in 54.5% of eyes, or 61.4% of patients. The area of intraretinal RPE migration on OCT always correlated to areas of pigment clumping on fundus photography. Conversely, all but 1 eye with RPE pigment clumping on fundus photography also had areas of intraretinal RPE migration on OCT. The high incidence of intraretinal RPE migration observed above areas of drusen suggests that drusen may play physical and catalytic roles in facilitating intraretinal RPE migration in dry AMD patients.National Institutes of Health (U.S.) (Contract RO1-EY11289-23)National Institutes of Health (U.S.) (Contract R01-EY13178-07)National Institutes of Health (U.S.) (Contract R01-EY013516-07)United States. Air Force Office of Scientific Research (FA9550-07-1-0101)United States. Air Force Office of Scientific Research (FA9550-07-1-0014

Assessment of Artifacts and Reproducibility across Spectral- and Time-Domain Optical Coherence Tomography Devices

Purpose To report the frequency of optical coherence tomography (OCT) scan artifacts and to compare macular thickness measurements, interscan reproducibility, and interdevice agreeability across 3 spectral-domain (SD) OCT (also known as Fourier domain; Cirrus HD-OCT, RTVue-100, and Topcon 3D-OCT 1000) devices and 1 time-domain (TD) OCT (Stratus OCT) device. Design Prospective, noncomparative, noninterventional case series. Participants Fifty-two patients seen at the New England Eye Center, Tufts Medical Center Retina Service, between February and August 2008. Methods Two scans were performed for each of the SD OCT protocols: Cirrus macular cube 512×128 (software version 3.0; Carl Zeiss Meditec, Inc., Dublin, CA), RTVue (E)MM5 and MM6 (software version 3.5; Optovue, Inc., Fremont, CA), Topcon 3D Macular and Radial (software version 2.12; Topcon, Inc., Paramus, NJ), in addition to 1 TD OCT scan via Stratus macular thickness protocol (software version 4.0; Carl Zeiss Meditec, Inc.). Scans were inspected for 6 types of OCT scan artifacts and were analyzed. Interscan reproducibility and interdevice agreeability were assessed by intraclass correlation coefficients (ICCs) and Bland-Altman plots, respectively. Main Outcome Measures Optical coherence tomography image artifacts, macular thickness, reproducibility, and agreeability. Results Time-domain OCT scans contained a significantly higher percentage of clinically significant improper central foveal thickness (IFT) after manual correction (11-μm change or more) compared with SD OCT scans. Cirrus HD-OCT had a significantly lower percentage of clinically significant IFT (11.1%) compared with the other SD OCT devices (Topcon 3D, 20.4%; Topcon Radial, 29.6%; RTVue (E)MM5, 42.6%; RTVue MM6, 24.1%; P = 0.001). All 3 SD OCT devices had central foveal subfield thicknesses that were significantly more than that of TD OCT after manual correction (P<0.0001). All 3 SD OCT devices demonstrated a high degree of reproducibility in the central foveal region (ICCs, 0.92–0.97). Bland-Altman plots showed low agreeability between TD and SD OCT scans. Conclusions Out of all OCT devices analyzed, cirrus HD-OCT scans exhibited the lowest occurrence of any artifacts (68.5%), IFT (40.7%), and clinically significant IFT (11.1%), whereas Stratus OCT scans exhibited the highest occurrence of clinically significant IFT. Further work on improving segmentation algorithm to decrease artifacts is warranted.Research to Prevent Blindness, Inc. (United States) (Challenge Grant)National Institutes of Health (U.S.) (Grant R01-EY11289-23)National Institutes of Health (U.S.) (Grant R01-EY13178-07)National Institutes of Health (U.S.) (Grant P30-EY008098)United States. Air Force Office of Scientific Research (Grant FA9550-07-1-0101)United States. Air Force Office of Scientific Research (Grant FA9550-07-1-0014

Surface Decorated Zn0.15Cd0.85S Nanoflowers with P25 for Enhanced Visible Light Driven Photocatalytic Degradation of Rh-B and Stability

Decoration of Zn0.15Cd0.85S nanoflowers with P25 for forming P25/Zn0.15Cd0.85S nanocomposite has been successfully synthesized with fine crystallinity by one-step low temperature hydrothermal method. Photocatalytic efficiency of the as-prepared P25/Zn0.15Cd0.85S for the degradation of Rh-B is evaluated under the visible light irradiation. The synthesized composite is completely characterized with XRD, FESEM, TEM, BET, and UV-vis DRS. TEM observations reveal that P25 is closely deposited on the Zn0.15Cd0.85S nanoflowers with maintaining its nanoflower morphology. The photocatalytic activity of the as-obtained photocatalyst shows that the P25/Zn0.15Cd0.85S exhibits very high catalytic activity for degradation of Rh-B under visible light irradiation due to an increasing of the active sites and enhancing the catalyst stability because of the minimum recombination of the photo-induced electrons and holes. Moreover, it is found that the nanocomposite retains its photocatalytic activity even after four cycles. In addition, to explain the mechanism of degradation, scavengers are used to confirm the reactive species. Photo-generated holes and ●OH play a significant role in the visible light of P25/Zn0.15Cd0.85S nanocomposite induced degradation system, but electrons play the most important role

Multiplicity of concentrating solutions for a class of magnetic Schrödinger-Poisson type equation

In this paper, we study the following nonlinear magnetic Schrödinger-Poisson type equatio