The limited role of NH2-terminal c-Jun phosphorylation in neuronal apoptosis: Identification of the nuclear pore complex as a potential target of the JNK pathway

c-Jun is induced in many neuronal death paradigms. A critical step in c-Jun regulation involves phosphorylation of Ser63/Ser73 located in the NH2-terminal transactivation domain. To determine the importance of this phosphorylation for neuronal apoptosis, we analyzed the sympathetic neurons of mice carrying a mutant c-Jun gene that lacks Ser63/Ser73 phosphorylation sites (jun aa). Trophic factor–deprivation or DNA damage–induced death was significantly delayed in jun aa/aa neurons. Neuronal c-Jun induction was only partially inhibited, demonstrating that phosphorylation of Ser63/73 is not required for c-Jun activation. The inductions of proapoptotic BH3-only proteins, Bim and PUMA/Bbc3, were delayed during neuronal apoptosis in mutant neurons. These results demonstrate that NH2-terminal c-Jun phosphorylation is important, but not necessary, for the induction of proapoptotic genes and neuronal apoptosis. Thus, additional JNK substrates may be critical for neuronal death. As potential mediators, we identified additional nuclear MLK/JNK substrates, including Nup214 subunit of the nuclear pore complex

Magnetic Hyperthermia in Y79 Retinoblastoma and ARPE19 Retinal Epithelial Cells: Tumor Selective Apoptotic Activity of Iron Oxide Nanoparticle

Purpose: To evaluate selective apoptosis of Y79 retinoblastoma versus ARPE-19 retinal pigment epithelial cells by using different doses of dextran-coated iron oxide nanoparticles (DCIONs) in a magnetic hyperthermia paradigm. Methods: Y79 and ARPE-19 cells were exposed to different concentrations of DCIONs, namely, 0.25, 0.5, 0.75, and 1 mg/ml. After 2 hours of incubation, cells were exposed to a magnetic field with a frequency of 250 kHz and an amplitude of 4 kA/m for 30 minutes to raise the cellular temperature between 42 and 46°C. Y79 and ARPE-19 cells incubated with DCION without magnetic field exposure were used as controls. Cell viability and apoptosis were assessed at 4, 24, and 72 hours after hyperthermia treatment. Results: At 4 hours following magnetic hyperthermia, cell death for Y79 cells was 1%, 8%, 17%, and 17% for 0.25, 0.5, 0.75 and 1 mg/ml of DCION, respectively. Cell death increased to 47%, 59%, 70%, and 75% at 24 hours and 16%, 45%, 50%, and 56% at 72 hours for 0.25, 0.5, 0.75, and 1 mg/ml of DCIONs, respectively. Magnetic hyperthermia did not have any significant toxic effects on ARPE-19 cells at all DCION concentrations, and minimal baseline cytotoxicity of DCIONs on Y79 and ARPE-19 cells was observed without magnetic field activation. Gene expression profiling showed that genes involved in FAS and tumor necrosis factor alpha signaling pathways were activated in Y79 cells following hyperthermia. Caspase 3/7 activity in Y79 cells increased following treatment, consistent with the activation of caspase-mediated apoptosis and loss of cell viability by magnetic hyperthermia. Conclusion: Magnetic hyperthermia using DCIONs selectively kills Y79 cells at 0.5 mg/ml or higher concentrations via the activation of apoptotic pathways. Translational Relevance: Magnetic hyperthermia using DCIONs might play a role in targeted management of retinoblastoma

Smartphone-based, rapid, wide-field fundus photography for diagnosis of pediatric retinal diseases

PurposeAn important, unmet clinical need is for cost-effective, reliable, easy-to-use, and portable retinal photography to evaluate preventable causes of vision loss in children. This study presents the feasibility of a novel smartphone-based retinal imaging device tailored to imaging the pediatric fundus.MethodsSeveral modifications for children were made to our previous device, including a child-friendly 3D printed housing of animals, attention-grabbing targets, enhanced image stitching, and video-recording capabilities. Retinal photographs were obtained in children undergoing routine dilated eye examination. Experienced masked retina-specialist graders determined photograph quality and made diagnoses based on the images, which were compared to the treating clinician's diagnosis.ResultsDilated fundus photographs were acquired in 43 patients with a mean age of 6.7 years. The diagnoses included retinoblastoma, Coats' disease, commotio retinae, and optic nerve hypoplasia, among others. Mean time to acquire five standard photographs totaling 90-degree field of vision was 2.3 ± 1.1 minutes. Patients rated their experience of image acquisition favorably, with a Likert score of 4.6 ± 0.8 out of 5. There was 96% agreement between image-based diagnosis and the treating clinician's diagnosis.ConclusionsWe report a handheld smartphone-based device with modifications tailored for wide-field fundus photography in pediatric patients that can rapidly acquire fundus photos while being well-tolerated.Translational relevanceAdvances in handheld smartphone-based fundus photography devices decrease the technical barrier for image acquisition in children and may potentially increase access to ophthalmic care in communities with limited resources

Growth Kinetics in Layer‐by‐Layer Assemblies of Organic Nanoparticles and Polyelectrolytes

The growth rates of layer‐by‐layer (LbL) assemblies of polyelectrolytes (PEs) with oppositely charged polystyrene (PS) nanoparticles (NPs) as a function of molecular weight (MW) of the PEs, ionic strength of the media, and NP size and charge are systematically investigated. To optimize LbL growth, the effects of suspension concentration, pH of the media, and deposition time on the growth rate of multilayers are assessed. Both linear and exponential growth behaviors are observed and, under optimal conditions, films of up to around 1 μm thick can readily be assembled after 10 or so bilayers have been deposited. For many of the cases studied, an intermediate MW of PE leads to the fastest film buildup, for both cationic poly(ethyleneimine) deposited alternately with anionic PS NPs and for anionic poly(acrylic acid) deposited alternately with cationic PS NPs. The existence of an optimal MW suggests that growth rate is determined by a balance of thermodynamic factors, including density of polymer bridges between particles, and kinetic factors, specifically the diffusivity of polymer in the film. The optimal MW, however, is very sensitive to the materials used. Moreover, depending on the MW of the PE, increasing salinity could increase or decrease the growth kinetics. Finally, the surface morphology of the films is characterized with AFM and SEM to reveal that the roughness increases less than linearly with film thickness.Growth factors: The growth rates of layer‐by‐layer (LbL) assemblies of polyelectrolytes (PEs) with oppositely charged polystyrene nanoparticles are systematically investigated. The molecular weight of a PE has a considerable effect on LbL film growth and its surface morphology (see figure).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135666/1/cphc201600789_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135666/2/cphc201600789-sup-0001-misc_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135666/3/cphc201600789.pd

First-in-Human Gene Therapy Trial of AAV8-hCARp.hCNGB3 in Adults and Children With CNGB3-associated Achromatopsia

Purpose: To assess the safety and efficacy of AAV8-hCARp.hCNGB3 in participants with CNGB3-associated achromatopsia (ACHM). Design: Prospective, phase 1/2 (NCT03001310), open-label, nonrandomized clinical trial. Methods: The study enrolled 23 adults and children with CNGB3-associated ACHM. In the dose-escalation phase, adult participants were administered 1 of 3 AAV8-hCARp.hCNGB3 dose levels in the worse-seeing eye (up to 0.5 mL). After a maximum tolerated dose was established in adults, an expansion phase was conducted in children ≥3 years old. All participants received topical and oral corticosteroids. Safety and efficacy parameters, including treatment-related adverse events and visual acuity, retinal sensitivity, color vision, and light sensitivity, were assessed for 6 months. Results: AAV8-hCARp.hCNGB3 (11 adults, 12 children) was safe and generally well tolerated. Intraocular inflammation occurred in 9 of 23 participants and was mainly mild or moderate in severity. Severe cases occurred primarily at the highest dose. Two events were considered serious and dose limiting. All intraocular inflammation resolved following topical and systemic steroids. There was no consistent pattern of change from baseline to week 24 for any efficacy assessment. However, favorable changes were observed for individual participants across several assessments, including color vision (n = 6/23), photoaversion (n = 11/20), and vision-related quality-of-life questionnaires (n = 21/23). Conclusions: AAV8-hCARp.hCNGB3 for CNGB3-associated ACHM demonstrated an acceptable safety and tolerability profile. Improvements in several efficacy parameters indicate that AAV8-hCARp.hCNGB3 gene therapy may provide benefit. These findings, with the development of additional sensitive and quantitative end points, support continued investigation.</p

First-in-Human Gene Therapy Trial of AAV8-hCARp.hCNGB3 in Adults and Children With CNGB3-associated Achromatopsia

PURPOSE: To assess the safety and efficacy of AAV8-hCARp.hCNGB3 in participants with CNGB3-associated achromatopsia (ACHM). DESIGN: Prospective, phase 1/2 (NCT03001310), open-label, nonrandomized clinical trial. METHODS: The study enrolled 23 adults and children with CNGB3-associated ACHM. In the dose-escalation phase, adult participants were administered 1 of 3 AAV8-hCARp.hCNGB3 dose levels in the worse-seeing eye (up to 0.5 mL). After a maximum tolerated dose was established in adults, an expansion phase was conducted in children ≥3 years old. All participants received topical and oral corticosteroids. Safety and efficacy parameters, including treatment-related adverse events and visual acuity, retinal sensitivity, color vision, and light sensitivity, were assessed for 6 months. RESULTS: AAV8-hCARp.hCNGB3 (11 adults, 12 children) was safe and generally well tolerated. Intraocular inflammation occurred in 9 of 23 participants and was mainly mild or moderate in severity. Severe cases occurred primarily at the highest dose. Two events were considered serious and dose limiting. All intraocular inflammation resolved following topical and systemic steroids. There was no consistent pattern of change from baseline to week 24 for any efficacy assessment. However, favorable changes were observed for individual participants across several assessments, including color vision (n = 6/23), photoaversion (n = 11/20), and vision-related quality-of-life questionnaires (n = 21/23). CONCLUSIONS: AAV8-hCARp.hCNGB3 for CNGB3-associated ACHM demonstrated an acceptable safety and tolerability profile. Improvements in several efficacy parameters indicate that AAV8-hCARp.hCNGB3 gene therapy may provide benefit. These findings, with the development of additional sensitive and quantitative end points, support continued investigation

Recovery of outer retina in acute idiopathic blind spot enlargement (AIBSE)

Purpose: To report the anatomic and functional recovery of the ellipsoid zone in a case of acute idiopathic blind spot enlargement (AIBSE), which was documented by serial high-resolution optical coherence tomography imaging. Observations: The patient's clinical presentation and follow up visits were documented via Humphrey's Visual Fields, fundus autofluorescence, and high resolution spectral domain optical coherence tomography (SD-OCT). At presentation, Humphrey's Visual Field testing showed an enlarged blind spot in the right eye. Fundus autofluorescence and optical coherence tomography showed an increased peripapillary autofluorescence and loss of the outer retinal layers, respectively. At 3 months a modest improvement in the visual field was observed. This improvement was stable at both the 7 and the 15 month follow up visits. SD-OCT corresponding to the areas of visual field improvement demonstrated recovery of the outer retina. Conclusion and importance: Serial OCT imaging demonstrated anatomic evidence of ellipsoid zone recovery in isolated AIBSE. Anatomic recovery was consistent with the functional gain detected by visual field improvement

The Atp1b3 gene for Na,K-ATPase β 3 subunit maps to mouse Chromosome 9, and a related gene, Atp1b3-rs, maps to mouse Chromosome 3

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42144/1/335-9-2-171_9n2p171.pd