Purpose:Light detection destroys the visual pigment. Its regeneration, necessary for the recovery of light sensitivity, is accomplished through the visual cycle. Release of all-trans retinal by the light-activated visual pigment and its reduction to all-trans retinol comprise the first steps of the visual cycle. In this study, we determined the kinetics of all-trans retinol formation in human rod and cone photoreceptors. Methods:Single living rod and cone photoreceptors were isolated from the retinas of human cadaver eyes (ages 21 to 90 years). Formation of all-trans retinol was measured by imaging its outer segment fluorescence (excitation, 360 nm; emission, >420 nm). The extent of conversion of released all-trans retinal to all-trans retinol was determined by measuring the fluorescence excited by 340 and 380 nm. Measurements were repeated with photoreceptors isolated from Macaca fascicularis retinas. Experiments were carried out at 37°C. Results:We found that ∼80% to 90% of all-trans retinal released by the light-activated pigment is converted to all-trans retinol, with a rate constant of 0.24 to 0.55 min-1 in human rods and ∼1.8 min-1 in human cones. In M. fascicularis rods and cones, the rate constants were 0.38 ± 0.08 min-1 and 4.0 ± 1.1 min-1, respectively. These kinetics are several times faster than those measured in other vertebrates. Interphotoreceptor retinoid-binding protein facilitated the removal of all-trans retinol from human rods. Conclusions:The first steps of the visual cycle in human photoreceptors are several times faster than in other vertebrates and in line with the rapid recovery of light sensitivity exhibited by the human visual system.

PURPOSE. Mutations in the gene that encodes the enzyme acid sphingomyelinase (ASMase) are associated with Niemann-Pick disease, a lysosomal storage disorder. Mice that lack ASMase (ASMase-/-) exhibit age-related retinal degeneration and large increases in accumulation of lipofuscin in the retinal pigment epithelium (RPE). We examined which lipid species accumulate in the retina and the RPE of ASMase-/-mice and whether the retinal degeneration is associated with impaired photoreceptor metabolism and retinyl chromophore processing. METHODS. NADPH availability and all-trans retinol formation after rhodopsin bleaching were measured in isolated single rod photoreceptors with fluorescence imaging; sphingolipid levels in retinas and RPEs were measured with LC/MS; relative abundances of different lipid species in different retinal layers were measured with MALDI imaging mass spectrometry. RESULTS. There was no detectable difference in the kinetics of all-trans retinol formation or the NADPH-generating capacity between ASMase-/- and wild-type mice. Sphingomyelin levels were much higher in the retinas and RPEs of ASMase-/- animals compared to wild type, but there were no significant differences for ceramides. There was a large increase in the abundance of bis(monoacylglycero)phosphates (BMPs) in ASMase-/-mice, indicative of lysosomal dysfunction, but no substantial changes were detected for the bis-retinoid A2E. CONCLUSIONS. Lysosomal dysfunction and retinal degeneration in ASMase-/-mice are not associated with defects in rod photoreceptor metabolism that affect all-trans retinol formation and availability of NADPH. Lysosomal dysfunction in ASMase-/-mice is not associated with bis-retinoid A2E accumulation.

Podocytes have a remarkable ability to recover from injury; however, little is known about the recovery mechanisms involved in this process. We recently showed that formoterol, a long-acting β2-adrenergic receptor (β2-AR) agonist, induced mitochondrial biogenesis (MB) in podocytes and led to renoprotection in mice. However, it is not clear whether this effect was mediated by formoterol acting through the β2-AR or if it occurred through “off-target” effects. We genetically deleted the β2-AR specifically in murine podocytes and used these mice to determine whether formoterol acting through the podocyte β2-AR alone is sufficient for recovery of renal filtration function following injury. The podocyte-specific β2-AR knockout mice (β2-ARfl/fl/PodCre) were generated by crossing β2-AR floxed mice with podocin Cre (B6.Cg-Tg(NPHS2-cre)295Lbh/J) mice. These mice were then subjected to both acute and chronic glomerular injury using nephrotoxic serum (NTS) and adriamycin (ADR), respectively. The extent of injury was evaluated by measuring albuminuria and histological and immunostaining analysis of the murine kidney sections. A similar level of injury was observed in β2-AR knockout and control mice; however, the β2-ARfl/fl/PodCre mice failed to recover in response to formoterol. Functional evaluation of the β2-ARfl/fl/PodCre mice following injury plus formoterol showed similar albuminuria and glomerular injury to control mice that were not treated with formoterol. These results indicate that the podocyte β2-AR is a critical component of the recovery mechanism and may serve as a novel therapeutic target for treating podocytopathies.

Supplemental Figure S3. A. Tumors harvested on treatment d 7 were assessed by IHC for Arg-1 and FoxP3. Representative images are shown. Original magnification, 400X. N = 5. B-C. RT-qPCR gene expression analysis of Ripk1 and Ripk3 (B) and Gas6 (C) from tumors treated with vehicle or lapatinib on treatment day 1, followed by 6 daily treatments with vehicle or BMS-777607. Tumors were harvested 24 h after final treatment. Individual data points represent the average value of 5 experimental replicates from RNA harvested from a single tumor. Midlines are the average ({plus minus} S.D.) of the biological replicates (N = 3-4 per group). P values calculated using Student's T-test.

4T1 mouse mammary tumors were grown orthotopically in athymic Balb/C (nu/nu) female mice. Tumor-bearing mice were randomized into treatment groups for intratumoral delivery of SLR20 (or OH-SLR20, or saline) on treatment days 1, 5, and 9. Tumors were measured throughout treatment (days 1-10) and for 5 days after treatment ceased (days 10-15). N = 7 per group.

Immunohistochemistry was used to measure CD45, F4,80, CD4, and CD8 in tumors harvested at day 5. Representative Images are shown. N = 5

4T1 cells were transfected with SLR20 or OH-SLR20. At 4 hours after transfection, 4T1 cells were washed 5 times, then cultured in serum-free media for 16 hours. 4T1-cultured supernatant was collected, passed through a 0.2ï­m filter, then added neat to cultures of Raw264.7 cells for 30 minutes. Western analysis of RAW264.7 whole cell lysates was used to measure P-STAT1. Cells were transfected, and after 16 h, total RNA was assessed by RT-qPCR to measure expression of the indicated gene.. Each point represents the average of three experimental replicates, N = 3. Midlines are average {plus minus} S.D. Student's T-test.

The effect of U46619 and CPI211 on migration and invasion of 4T1 and MDA-MB-231 tumor cells through Matrigel-coated transwells was assessed. Tumor cells were seeded in the upper chambers of a Matrigel-coated transwell insert in serum-free media. Cell migration towards 1% serum in the lower chamber was assessed by crystal violet staining of the lower side of the transwell filter. Digital images of stained transwell filters were used to count the number of migrated cells. Representative images are shown (A). Each data point shown (B) represents the number of cells migrating to the lower side of the filter. Midlines are the average {plus minus} S.D., N = 4 (4T1) and 5 (MDA-MB-231), each assessed in triplicate.

Supplemental Figure S6. Tumors harvested on treatment d 7 were assessed by IHC for FoxP3 and Arg-1. Representative images are shown. Original magnification = 400X. Asterisks represent areas of acellular debris. Black arrows indicate tumor infiltrating lymphocytes (TILs). Yellow arrows indicate hyper-condensed nuclei characteristic of apoptotic bodies / apoptotic debris.

A. Schematic of treatment strategy for intra-tumoral nanoparticle delivery of SLR20 (or OH-SLR20) to WT Balb/C mice harboring 4T1 mammary tumors. Saline was delivered intratumorally as a control. Tumors were measured throughout treatment . B. Tumor volume was measured beginning at treatment day 0. N = 7-8 per group.

Chemical structure of CPI211 is shown in Panel A. Western analysis of 293T cells expressing lentiviral empty vector, TPr-WT, or TPr-T399A s shown in Panel B. Antibodies used are shown to the left of each panel.

S3. Representative images of cells grown in 3D Matrigel for 14D and treated with ABT-263 (1.0 uM). S4. Whole cell lysates from cells treated with ABT-263 (1.0uM) for 0=24 hours.

Supplemental Figure S1. Tumors harvested on treatment d 1 and d 7 were assessed by IHC for P-Neu Y1248. Representative images are shown. Original magnification, 400X. N = 5. Histological analysis of H&E-stained tumor sections. Tumors were collected on treatment day 2 (i.e., 1 day after treatment {plus minus} lapatinib). Representative images are shown. N = 5. Original magnification was 400X. Tumors harvested on treatment d 2 were assessed by IHC for CD3 and FoxP3. Representative images are shown. Original magnification, 400X. N = 5. Tumors harvested on treatment d 2 were assessed by IHC for CD3 and FoxP3. Representative images are shown. Original magnification, 400X. N = 5. RT-qPCR analysis of tumor RNA harvested at treatment d 7 measuring relative levels of indicated gene transcripts. Values were calculated using the ddCT method. Each data point represents the average value of 5 technical replicates, N = 5 tumors. For each transcript, values were corrected for the average value measured in vehicle-treated samples. Student's T-test.

Supplemental Figure S5. Decreased tumor cellularity of tumors treated with the combination of lapatinib + BMS-777607 + epacadostat. Low power images of H&E-stained sections of tumors collected at treatment day 7. Arrows point to tumor cells, which stain darker purple, and which are evident as solid sheets in vehicle-treated samples, but only as pockets in the samples treated with lapatinib + BMS-777607 + Epacadostat. Asterisks represent areas of acellular debris.

Supplemental Figure S7. MMTV-Neu tumors were treated lapatinib on days 1 and 7, and with BMS-777607 on days 14. Average tumor volume is shown.

A 74-year-old man with a history of primary open-angle glaucoma and trabeculectomy presented with a giant pigmented superior fornix mass (A). Vision was hand motion with normal intraocular pressure. Examination showed pigmented satellite lesions (B), an irregularly thickened iris (C), and anterior chamber cell. Exenteration was performed. Pathology showed primary acquired melanosis with atypia at the trabeculectomy site and melanoma located within the conjunctival substantia propria, cornea, angle structures, and uveal tract (D, arrows). The tumor exhibited a mutation in NRAS but no alterations in GNAQ or GNA11 genes. These findings confirmed a conjunctival melanoma had likely seeded the intraocular structures through the previous trabeculectomy (Magnified version of Fig A-D is available online at www.aaojournal.org).

S1. CCLE-curated cancer cells were grouped according to tumor type and assessed for miRNA expression for BCL2 and BCL2L1 (Bcl-xL) using cBio portal (www.cbio.org). S2. Whole cell lysates form a panel of breast cancer cell lines were assessed by western analysis (BL = Basal-like).

Supplemental Figure S2. Schematic representation of treatment groups and experimental timeline to measure the impact of PtdSer liposomes on the TME of 4T1 mouse mammary tumors IHC to detect CD3, and FoxP3 in tumors collected on treatment day 7. Quantitation of the number of positive cells per 200X field is shown. Each data point is the average of 5 random fields per tumor, midlines are the average of N = 3 samples, {plus minus}S.D. P values, Student's unpaired 2-tailed T-test.

Western analysis of whole cel lysates harvested from BT474, MCF7, and MDA-MB-361 cells using antibodies indicated at the left of each panel. Western analysis of whole cell lysates harvested from MDA-MB-361 cells tranfected with SLR20 or OH-SLR20 at 12 hours after transfection. Immunohistochemistry was used to measure RIG-I and P-STAT1 in tumors harvested at day 5. Representative Images are shown. N = 5.

Supplemental Figure S8. MMTV-Neu tumors were measured on treatment day 1 and again on treatment day 29. measurements from each tumor is represented by a datapoint. The line connects measurements from a single tumor on day 1 to the measurement of the same tumor on day 29. Vehicle-treated tumors are shown in gray. A. Vehicle-treated tumors are compared to tumors treated with lapatinib (shown in black). B. Vehicle-treated tumors are compared to tumors treated with BMS-777607 + Epacadostat (B/E, shown in red). C. Vehicle-treated tumors are compared to tumors treated with Lapatinib + BMS-777607 + Epacadostat (L/B/E, shown in blue). D. Vehicle-treated tumors are compared to all remaining treatment groups: lapatinib-treated tumors in black, B/E-treated tumors in red, and L/B/E-treated tumors in blue.