Sigma 1 receptor (Sig1R), a modulator of cell survival, has emerged as a novel target for retinal degenerative disease. Studies have shown that activation of Sig1R, using the high affinity ligand (+)-pentazocine ((+)-PTZ), improves cone function in a severe retinopathy model. The rescue is accompanied by normalization of levels of NRF2, a key transcription factor that regulates the antioxidant response. The interaction of Sig1R with a number of proteins has been investigated; whether it interacts with NRF2, however, is not known. We used co-immunoprecipitation (co-IP), proximity ligation assay (PLA), and electron microscopy (EM) immunodetection methods to investigate this question in the 661W cone photoreceptor cell line. For co-IP experiments, immune complexes were precipitated by protein A/G agarose beads and immunodetected using anti-NRF2 antibody. For PLA, cells were incubated with anti-Sig1R polyclonal and anti-NRF2 monoclonal antibodies, then subsequently with (−)-mouse and (+)-rabbit PLA probes. For EM analysis, immuno-EM gold labeling was performed using nanogold-enhanced labeling with anti-NRF2 and anti-Sig1R antibodies, and data were confirmed using colloidal gold labeling. The co-IP experiment suggested that NRF2 was bound in a complex with Sig1R. The PLA assays detected abundant orange fluorescence in cones, indicating that Sig1R and NRF2 were within 40 nm of each other. EM immunodetection confirmed co-localization of Sig1R with NRF2 in cells and in mouse retinal tissue. This study is the first to report co-localization of Sig1R-NRF2 and supports earlier studies implicating modulation of NRF2 as a mechanism by which Sig1R mediates retinal neuroprotection.

(1) Background: caspase-12 is activated during cytomegalovirus retinitis, although its role is presently unclear. (2) Methods: caspase-12^−/− (KO) or caspase-12^+/+ (WT) mice were immunosup eyes were analyzed by plaque assay, TUNEL assay, immunohistochemical staining, western blotting, and real-time PCR. (3) Results: increased retinitis and a more extensive virus spread were detected in the retina of infected eyes of KO mice compared to WT mice at day 14 p.i. Compared to MCMV injected WT eyes, mRNA levels of interferons α, β and γ were significantly reduced in the neural retina of MCMV-infected KO eyes at day 14 p.i. Although similar numbers of MCMV infected cells, similar virus titers and similar numbers of TUNEL-staining cells were detected in injected eyes of both KO and WT mice at days 7 and 10 p.i., significantly lower amounts of cleaved caspase-3 and p53 protein were detected in infected eyes of KO mice at both time points. (4) Conclusions: caspase-12 contributes to caspase-3-dependent and independent retinal bystander cell death during MCMV retinitis and may also play an important role in innate immunity against virus infection of the retina.

Our previous studies have shown that systemic neonatal murine cytomegalovirus (MCMV) infection of BALB/c mice spread to the eye with subsequent establishment of latency in choroid/RPE. In this study, RNA sequencing (RNA-Seq) analysis was used to determine the molecular genetic changes and pathways affected by ocular MCMV latency. MCMV (50 pfu per mouse) or medium as control were injected intra-peritoneally (i.p.) into BALB/c mice at <3 days after birth. At 18 months post injection, the mice were euthanized, and the eyes were collected and prepared for RNA-Seq. Compared to three uninfected control eyes, we identified 321 differentially expressed genes (DEGs) in six infected eyes. Using the QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA), we identified 17 affected canonical pathways, 10 of which function in neuroretinal signaling, with the majority of DEGs being downregulated, while 7 pathways function in upregulated immune/inflammatory responses. Retinal and epithelial cell death pathways involving both apoptosis and necroptosis were also activated. MCMV ocular latency is associated with upregulation of immune and inflammatory responses and downregulation of multiple neuroretinal signaling pathways. Cell death signaling pathways are also activated and contribute to the degeneration of photoreceptors, RPE, and choroidal capillaries.

Poor aqueous solubility remains a major barrier to the development, repurposing, and improved delivery of many clinically important small-molecule drugs. This problem is especially important for Biopharmaceutics Classification System (BCS) Class II drugs, in which intestinal permeability may be acceptable but dissolution and aqueous formulation compatibility limit absorption, formulation flexibility, and translational testing. Current formulation-enabling strategies, including solid dispersions, nanosuspensions, nanocrystals, lipid-based systems, cyclodextrin complexes, self-emulsifying systems, and polymer- or surfactant-stabilized particles, can improve drug dissolution and exposure, but these approaches often require drug-specific formulation optimization, additional carriers, multi-step processing, or prolonged development. In this study, we evaluated Facilitated Self-Assembling Technology (FAST) as a rapid carrier-free aqueous nanoformulation platform for eleven poorly water-soluble small-molecule drugs and one inorganic dental material. Ten selected compounds were representative BCS Class II drugs: celecoxib, itraconazole, rifampin, sorafenib, spironolactone, carvedilol, ezetimibe, lurasidone, rivaroxaban, and fenofibrate. Propofol was included as an additional clinically important hydrophobic drug to introduce the concept that FAST may enable “me-better” formulation opportunities for approved drugs with route-specific delivery limitations. Omyadent HA100 hydroxyapatite was included as a proof-of-concept inorganic oral-care material because hydroxyapatite is widely used as a biomimetic dental mineral and nanoscale hydroxyapatite has been investigated for enamel/dentin remineralization and dentin tubule occlusion; the HA100 experiment was designed to evaluate whether FAST could generate nanoscale mineral clusters under mild aqueous conditions. Using FAST, the selected drugs were converted into aqueous nanoscale dispersions and characterized by dynamic light scattering (DLS)/ZetaView analysis. In the initial ten-drug dataset, all formulations were generated and characterized within a same-day workflow, with particle sizes primarily in the nanoscale range and negative zeta potentials. Propofol, itraconazole, and rifampin were evaluated using the same analytical scheme, including particle size, particle concentration, zeta potential, visual dispersion stability, and TEM imaging. Omyadent HA100 was evaluated by DLS/ZetaView and TEM as an inorganic proof-of-concept material. TEM imaging of itraconazole revealed mixed morphology, including rod- or needle-like crystalline/nanocrystalline structures and rounded nanoscale particles, while rifampin showed heterogeneous rounded/oval particles and irregular nanoscale clusters in a clear brown/amber suspension. Together, these studies test whether FAST can rapidly generate carrier-free aqueous nanoformulations across chemically diverse poorly soluble drugs and a nanoscale hydroxyapatite material. The current study does not claim improved dissolution, pharmacokinetics, bioavailability, or therapeutic efficacy for each drug. Instead, it establishes a formulation-feasibility framework. If further studies confirm improved dissolution, stability, drug loading, route-specific delivery, pharmacokinetics, and safety, FAST may provide a rapid self-assembly platform for early formulation screening, drug repurposing, AI-assisted drug discovery, and me-better development of approved drugs with formulation-limited clinical performance.

Poor aqueous solubility remains a major challenge in formulating BCS Class II and IV drugs, while therapeutic peptides face additional challenges in stability and biological delivery. In the present study, we evaluated Facilitated Self-Assembling Technology (FAST) for the rapid preparation of aqueous nanosuspensions of 11 clinically relevant small-molecule drugs and two lipidated peptides, semaglutide and retatrutide. The compounds represent diverse structural classes and therapeutic uses, including targeted anticancer agents, immunosuppressants, anti-inflammatory drugs, and incretin-based peptides. Nanoparticle tracking analysis detected FAST-generated nanoscale particle populations in all 13 preparations, with number-weighted mean diameters from 126.7 to 219.1 nm and particle concentrations from 9.0 × 10⁷ to 5.4 × 10⁹ particles/mL. Semaglutide and retatrutide yielded mean diameters of 126.7 ± 55.6 and 163.0 ± 64.6 nm, respectively. All preparations exhibited negative zeta potentials. Paired-vial photographs showed similar aqueous appearances after more than 30 days at room temperature, except a slightly darker color in the venetoclax preparation. Transmission electron microscopy demonstrated predominantly rounded nanoscale particles in representative adagrasib and cyclosporine preparations. X-ray diffraction profiles of six selected FAST-generated BCS Class II/IV drug formulations exhibited broad, diffuse features near 20° 2θ, suggesting low apparent crystalline order and amorphous-like characteristics, which may favor improved dissolution of poorly water-soluble drugs. Taken together, these results support our hypothesis that FAST can generate aqueous nanoparticle preparations from clinically important compounds with widely different molecular structures. The extension to lipidated peptides suggest opportunities for future development of alternative peptide formulations and delivery systems.

(1) Background: Retinal degeneration develops upon caspase-12 activation in aged BALB/c mice following systemic neonatal infection. (2) Methods: MCMV or medium was injected intraperitoneally (i.p.) into caspase-12^−/− and caspase-12^+/+ mice (on BALB/c background) at <3 days after birth. At 8 and 12 months post infection (p.i.), eyes were analyzed by SD-OCT before eyes and extraocular tissues were collected and analyzed by plaque assay, H&E staining, TUNEL assay, Western blot and real-time RT-PCR. (3) Results: Virus DNA, but not replicating virus, was present in eyes and extraocular tissues at 8 and 12 months p.i. Several MCMV genes were expressed in eyes of both MCMV-infected caspase-12^−/− and caspase-12^+/+ mice, while mean retinal thickness was significantly higher in MCMV latently infected aged caspase-12^−/− mice compared to age-matched infected caspase-12^+/+ mice. Although similar levels of cleaved caspase-1 were detected in eyes of both infected caspase-12^−/− and control mice, significantly higher levels of activated NF-κB, cleaved caspase-8, MLKL, p-RIP3 and p53 were observed in eyes of infected caspase-12^+/+ mice compared to eyes of infected caspase-12^−/− mice. (4) Conclusions: Our results suggest that caspase-12 contributes to retinal degeneration during MCMV ocular latency via multiple pathways including apoptosis and necroptosis.