snRNA-seq of human cutaneous neurofibromas before and after selumetinib treatment implicates role of altered Schwann cell states, inter-cellular signaling, and extracellular matrix in treatment response.

Neurofibromatosis Type 1 (NF1) is caused by loss of function variants in the NF1 gene. Most patients with NF1 develop skin lesions called cutaneous neurofibromas (cNFs). Currently the only approved therapeutic for NF1 is selumetinib, a mitogen -activated protein kinase (MEK) inhibitor. The purpose of this study was to analyze the transcriptome of cNF tumors before and on selumetinib treatment to understand both tumor composition and response. We obtained biopsy sets of tumors both pre- and on- selumetinib treatment from the same individuals and were able to collect sets from four separate individuals. We sequenced mRNA from 5844 nuclei and identified 30,442 genes in the untreated group and sequenced 5701 nuclei and identified 30,127 genes in the selumetinib treated group. We identified and quantified distinct populations of cells (Schwann cells, fibroblasts, pericytes, myeloid cells, melanocytes, keratinocytes, and two populations of endothelial cells). While we anticipated that cell proportions might change with treatment, we did not identify any one cell population that changed significantly, likely due to an inherent level of variability between tumors. We also evaluated differential gene expression based on drug treatment in each cell type. Ingenuity pathway analysis (IPA) was also used to identify pathways that differ on treatment. As anticipated, we identified a significant decrease in ERK/MAPK signaling in cells including Schwann cells but most specifically in myeloid cells. Interestingly, there is a significant decrease in opioid signaling in myeloid and endothelial cells; this downward trend is also observed in Schwann cells and fibroblasts. Cell communication was assessed by RNA velocity, Scriabin, and CellChat analyses which indicated that Schwann cells and fibroblasts have dramatically altered cell states defined by specific gene expression signatures following treatment (RNA velocity). There are dramatic changes in receptor-ligand pairs following treatment (Scriabin), and robust intercellular signaling between virtually all cell types associated with extracellular matrix (ECM) pathways (Collagen, Laminin, Fibronectin, and Nectin) is downregulated after treatment. These response specific gene signatures and interaction pathways could provide clues for understanding treatment outcomes or inform future therapies.

Enhanced thermophilic high-solids anaerobic digestion of organic fraction of municipal solid waste with spatial separation from conductive materials in a single reactor volume.

Despite benefits such as lower water and working volume requirements, thermophilic high solids anaerobic digestion (THSAD) often fails due to the rapid build-up of volatile fatty acids (VFAs) and the associated drop in pH. Use of conductive materials (CM) can promote THSAD through stimulation of direct interspecies electron transfer (DIET), while the need for their constant dosing due to poor separation from effluent impairs economic feasibility. This study used an approach of spatially separating magnetite and granular activated carbon (GAC) from the organic fraction of municipal solid waste (OFMSW) in a single reactor for THSAD. GAC and magnetite addition could both mitigate the severe inhibition of methanogenesis after VFAs build-up to ∼28-30 g/L, while negligible methane production was observed in the control group. The highest methane yield (286 mL CH4/g volatile solids (VS)) was achieved in magnetite-added reactors, while the highest maximum CH4 production rates (26.38 mL CH4/g VS/d) and lowest lag-phase (2.83 days) were obtained in GAC-added reactors. The enrichment of GAC and magnetite biofilms with various syntrophic and potentially electroactive microbial groups (Ruminiclostridium 1, Clostridia MBA03, Defluviitoga, Lentimicrobiaceae) in different relative abundances indicates the existence of specific preferences of these groups for the nature of CM. According to predicted basic metabolic functions, CM can enhance cellular processes and signals, lipid transport and metabolism, and methane metabolism, resulting in improved methane production. Rearrangement of metabolic pathways, formation of pili-like structures, enrichment of biofilms with electroactive groups and a significant improvement in THSAD performance was attributed to the enhancement of the DIET pathway. Promising results obtained in this work due to the spatial separation of the bulk OFMSW and CM can be useful for modeling larger-scale THSAD systems with better recovery of CM and cost-effectiveness.

Sustained neurotrophic factor cotreatment enhances donor and host retinal ganglion cell survival in mice.

Retinal ganglion cells (RGCs) lack regenerative capacity in mammals, and their degeneration in glaucoma leads to irreversible blindness. The transplantation of stem cell-derived RGCs lacks clinically relevant effect due to insufficient survival and integration of donor cells. We hypothesize that the retinal microenvironment plays a critical role in this process, and we can engineer a more acceptable setting for transplantation. Since the adult mammalian retina does not have regenerative capacity, we turned to the developing human retina to reconstruct cell-cell interactions at a single-cell level. We established a human fetal retina atlas by integrating currently available single-cell RNA sequencing datasets of human fetal retinas into a unified resource. We align RGC transcriptomes in pseudotime to map RGC developmental fate trajectories against the broader timeline of retinal development. Through this analysis, we identified brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) as key factors in RGC survival, highly expressed during fetal development but significantly reduced in adulthood despite the persistence of their receptors. To demonstrate the practical application of these findings, we show that using a slow-release formulation of BDNF and GDNF enhances RGC differentiation, survival, and function in vitro and improves RGC transplantation outcomes in a mouse model. BNDF/GDNF co-treatment not only increased survival and coverage of donor RGCs within the retina but also showed neuroprotective effects on host RGCs, preserving retinal function in a model of optic neuropathy. Altogether, our findings suggest that manipulating the retinal microenvironment with slow-release neurotrophic factors holds promise in regenerative medicine for treating glaucoma and other optic neuropathies. This approach not only improves donor cell survival and integration but also provides a neuroprotective benefit to host cells, indicating a significant advancement for glaucoma therapies.

Photoreceptors inhibit pathological retinal angiogenesis through transcriptional regulation of Adam17 via c-Fos.

Pathological retinal angiogenesis profoundly impacts visual function in vascular eye diseases, such as retinopathy of prematurity (ROP) in preterm infants and age-related macular degeneration in the elderly. While the involvement of photoreceptors in these diseases is recognized, the underlying mechanisms remain unclear. This study delved into the pivotal role of photoreceptors in regulating abnormal retinal blood vessel growth using an oxygen-induced retinopathy (OIR) mouse model through the c-Fos/A disintegrin and metalloprotease 17 (Adam17) axis. Our findings revealed a significant induction of c-Fos expression in rod photoreceptors, and c-Fos depletion in these cells inhibited pathological neovascularization and reduced blood vessel leakage in the OIR mouse model. Mechanistically, c-Fos directly regulated the transcription of Adam17 a shedding protease responsible for the production of bioactive molecules involved in inflammation, angiogenesis, and cell adhesion and migration. Furthermore, we demonstrated the therapeutic potential by using an adeno-associated virus carrying a rod photoreceptor-specific short hairpin RNA against c-fos which effectively mitigated abnormal retinal blood vessel overgrowth, restored retinal thickness, and improved electroretinographic (ERG) responses. In conclusion, this study highlights the significance of photoreceptor c-Fos in ROP pathology, offering a novel perspective for the treatment of this disease.

SOCS3 regulates pathological retinal angiogenesis through modulating SPP1 expression in microglia and macrophages.

Pathological ocular angiogenesis has long been associated with myeloid cell activation. However, the precise cellular and molecular mechanisms governing the intricate crosstalk between the immune system and vascular changes during ocular neovascularization formation remain elusive. In this study, we demonstrated that the absence of the suppressor of cytokine signaling 3 (SOCS3) in myeloid cells led to a substantial accumulation of microglia and macrophage subsets during the neovascularization process. Our single-cell RNA sequencing data analysis revealed a remarkable increase in the expression of the secreted phosphoprotein 1 (Spp1) gene within these microglia and macrophages, identifying subsets of Spp1-expressing microglia and macrophages during neovascularization formation in angiogenesis mouse models. Notably, the number of Spp1-expressing microglia and macrophages exhibited further elevation during neovascularization in mice lacking myeloid SOCS3. Moreover, our investigation unveiled the Spp1 gene as a direct transcriptional target gene of signal transducer and activator of transcription 3. Importantly, pharmaceutical activation of SOCS3 or blocking of SPP1 resulted in a significant reduction in pathological neovascularization. In conclusion, our study highlights the pivotal role of the SOCS3/STAT3/SPP1 axis in the regulation of pathological retinal angiogenesis.