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Uncovering the tissue molecular architecture at single-cell resolution could help better understand organisms’ biological and pathological processes. However, bulk RNA-seq can only measure gene expression in cell mixtures, without revealing the transcriptional heterogeneity and spatial patterns of single cells. Herein, we introduce Bulk2Space (https://github.com/ZJUFanLab/bulk2space), a deep learning framework-based spatial deconvolution algorithm that can simultaneously disclose the spatial and cellular heterogeneity of bulk RNA-seq data using existing single-cell and spatial transcriptomics references. The use of bulk transcriptomics to validate Bulk2Space unveils, in particular, the spatial variance of immune cells in different tumor regions, the molecular and spatial heterogeneity of tissues during inflammation-induced tumorigenesis, and spatial patterns of novel genes in different cell types. Moreover, Bulk2Space is utilized to perform spatial deconvolution analysis on bulk transcriptome data from two different mouse brain regions derived from our in-house developed sequencing approach termed Spatial-seq. We have not only reconstructed the hierarchical structure of the mouse isocortex but also further annotated cell types that were not identified by original methods in the mouse hypothalamus.

Purpose Retinitis pigmentosa GTPase regulator (RPGR)-related X-linked retinitis pigmentosa is associated with one of the most severe phenotypes among inherited retinal disease. The aim of this study was to investigate Clustered Regularly Interspaced Short Palindromic Repeat/Cas9-mediated gene editing therapy in a mouse model of Rpgr. Methods The Rpgr-/yCas9+/WT male mice were used for this study. At 6 months of age, they received a single subretinal injection of adeno-associated virus vectors carrying sgRNA and donor template separately, and therapeutic effect was examined after 1, 6, and 12 months. Results Rpgr knockout mouse showed slow but progressive age-related retinal degeneration, which emulates the disease occurring in humans. Significant photoreceptor preservation was observed in the treated part of the retina, in sharp contrast to the untreated part of the retina in the same eye after 6 and 12 months. It was surprising that precise modification at the target locus as demonstrated by genomic DNA sequencing in the post-mitotic photoreceptor was observed. Moreover, the therapeutic effect lasts for up to 12 months and no off-target effects were shown. Conclusions Our study strongly demonstrates that gene editing therapy is a promising therapeutic strategy to treat inherited retinal degeneration.

Osteoarthritis (OA) is a chronic arthritic disease that causes the overproduction of inflammatory factors such as nitric oxide (NO). This study develops a NO nanosensor to predict the OA development. The nanosensor is synthesized by encapsulating the NO sensing molecules (i.e., 4-amino-5-methylamino-2',7'-difluorofluorescein Diaminofluorescein-FM (DAF-FM)) within the biodegradable poly(lactic-co-glycolic acid) nanoparticles. In vitro, the nanosensor allows the monitoring of the NO release in interleukin-1β-stimulated chondrocytes and the alleviated effect of NG-monomethyl-l-arginine (a NO inhibitor) and andrographolide (an anti-inflammatory agent). In the rat OA model, it permits the quantification of NO level in joint fluid. The proposed NO nanosensor may facilitate a noninvasive and real-time evaluation of the OA development.

Neuroinflammation has been implicated in the mechanism underlying the progression of neurodegeneration and infectious neuropathology. Growing evidence suggest that hydroxytyrosol (3,4-dihydroxyphenil-ethanol, HT), one of the main polyphenols presented in extra virgin olive oil (EVOO), has shown potential anti-inflammatory and neuroprotective effects. However, the potential anti-neuroinflammation activity and underlying mechanism of HT remain poorly understood. The present study aimed to investigate the effects of HT on lipopolysaccharide (LPS)-induced inflammation in both in vitro and in vivo models and the associated molecular mechanism. Our results revealed that HT significantly reduced the production of pro-inflammatory mediators in BV2 microglia and primary microglia cells. Phenotypic analysis showed that HT significantly reduced M1 marker CD86 expression and increased M2 marker CD206 expression. In addition, HT significantly decreased the levels of phospho-NF-κB p65 and phospho-extracellular signal-regulated kinase (ERK) in a dose-dependent manner. Moreover, HT suppressed the LPS-induced Toll like receptor 4 (TLR4) in BV2 microglia. In vivo administration of HT following LPS injection significantly reduced some proinflammatory mediator levels and microglia/astrocyte activation in the brain. Together, these results suggest that HT suppressed the LPS-induced neuroinflammatory responses via modulation of microglia M1/M2 polarization and downregulation of TLR-4 mediated NF-κB activation and ERK signaling pathway.

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous disorder characterized by night blindness, visual field constriction, and severely reduced visual acuity. Despite a number of genes being implicated in RP pathogenesis, the genetic etiology of the disease remains unknown in many patients. In this study, our aim was to identify the disease-causing mutation of a large Chinese family with autosomal dominant RP (adRP). Targeted exon capture sequencing was initially performed to screen mutations in known disease-causing genes, followed by exome sequencing. In doing so, a heterozygous mutation in ADIPOR1 (c.929A > G) that results in an amino acid substitution (p.Y310C) was identified to co-segregate with the disease phenotype in this family. Adipor1 is wildly expressed throughout the body, but appears to be enriched in the photoreceptor inner and outer segments. The p.Y310C mutation, predicted to affect the structure and function of the protein, was confirmed to affect protein folding and its subcellular localization in vitro. In addition, knockdown of adipor1 expression in a zebrafish model with morpholino (MO) preferentially reduced the number of rod photoreceptors, with no effect on the number of cones, a phenotype that is characteristic of RP. Furthermore, the knockdown phenotype was partially rescued by injecting wild-type, but not mutant, human ADIPOR1 mRNA. We conclude that ADIPOR1 is a novel adRP-causing gene and plays an important role in rod development and maintenance.

Kainic acid (KA) administration is known to cause seizures and neuronal death in the hippocampus. High-frequency stimulation (HFS) of the hippocampus can be a promising method in the treatment of epilepsy while the mechanism of action is unknown yet. It remains unknown whether HFS is neuroprotective for hippocampal neurons following KA-induced seizures in macaques, although HFS has neuroprotective effects in animal models of Parkinson's disease. We therefore examined the effects of HFS on KA-induced seizures and neuronal survival in macaque's hippocampus. Seizure frequency following KA that led to seizures in macaques was strongly reduced by HFS of the hippocampus. In addition, administration of KA led to marked neuronal apoptosis in the hippocampus, accompanied by increased levels of Bax, activated caspase-3 and decreased levels of Bcl-2. HFS was found to attenuate changes in apoptosis-related proteins and robustly decreased neuronal loss following KA administration. These data indicate that hippocampal HFS can protect hippocampal neurons against KA neurotoxicity, and that HFS neuroprotection is likely to operate with inhibition of apoptosis.

Viili has been traditionally regarded as healthy food; viili exopolysaccharides (VEPS) function as antioxidants, but the molecular and cellular mechanisms, especially its immune functions, remain largely unclear. To assess VEPS's immunological roles, VEPS were separated by Sevage's method and purified by anion exchange chromatography. Cell proliferation, phagocytosis, releases of nitric oxide (NO), interleukin (IL)-1β, and IL-6, the inducible nitric oxide synthase (iNOS) gene expression by reverse transcription polymerase chain reaction (RT-PCR) and iNOS protein by Western blotting, and morphology by scanning electron microscopy in lipopolysaccharides (LPS)/VEPS-stimulated and non-stimulated RAW264.7 macrophages were analyzed. VEPS increased cell proliferation at 50-200 μg/mL. The uptake of neutral red for the indication of phagocytosis and releases of NO, IL-6, and IL-1β were enhanced after exposure to LPS and VEPS. Gene expressions of iNOS, IL-6, and IL-1β and protein expressions of iNOS were increased with VEPS. The RAW264.7 cell treated with VEPS became flattened, a strong indication of the activation of macrophages. We concluded that VEPS promoted the activation of macrophages in which NO, IL-6, and IL-1β were involved; the release of NO and other cytokines may eventually activate lymphocytes, increasing nonspecific (innate) and specific immunity in humans.

The Lewy body is a pathological hallmark of Parkinson's disease. It has been revealed that the Lewy body contains nitrated alpha-synuclein which is prone to oligomerization. We tested the hypothesis that aging may enhance nitration of alpha-synuclein due to an exaggerated neuroinflammatory reaction such as an excessive induction of the inducible nitric oxide synthase, which occurs post-intrapallidal lipopolysaccharide (LPS) injection. Here, we show microglia activation and proinflammatory cytokine expression are more evident in the substantia nigra of elderly rats following intrapallidal LPS. In addition, greater nitration of proteins like alpha-synuclein occurs in the substantia nigra of 16-month-old rats versus 3-month-old rats, which is accompanied by a higher expression level of inducible nitric oxide synthase. These results imply that an exaggerated neuroinflammatory response that occurs with aging might be involved in the increase in prevalence of neurodegenerative diseases like Parkinson's disease.

The motor behavior and levels of dopamine and its metabolites in the striatum were studied in rats that received a unilateral injection of 6-OHDA and underwent grafting of rat-derived primary fibroblasts that had been genetically modified to express lacZ and human glial cell line-derived neurotrophic factor (GDNF). Rotation behavior tests were performed each week and striatal levels of DA and its metabolites were measured every 4 weeks after grafting of fibroblasts that expressed lacZ, with or without additional transfection of the GDNF transgene. Rats grafted with GDNF-producing fibroblasts showed a significant improvement in motor behavior as determined by the rotation test, with a less pronounced reduction in the levels of dopamine and its metabolites in the striatum as compared with those in the control animals or brain parts. In addition, there was a lower decrease in the number of TH immunoreactive neurons in the substantia nigra ipsilateral to the lesion in rats with GDNF-producing fibroblasts than in rats with lacZ-expressing fibroblasts. These results support the notion that intracerebral grafting of fibroblasts that express GDNF is a potentially useful therapeutic strategy for treating Parkinson's disease.

Functional imaging of the non-human primate brain in awake animals is now feasible because of recent methodological advances. Here we detail our procedures for conducting functional MRI (fMRI) studies in rhesus monkeys. Our emphasis has been on analyzing drug-evoked responses within and across test groups, meaning that techniques have had to be developed for training and testing relatively large groups of animals. Group size is important as unbiased estimates are best derived from analyzing responses in multiple animals with replicate scans per animal due to partial volume errors in evaluating small brain regions and motion artifacts during scanning. While the procedures presented here were developed for mapping responses obtained from stimulating dopaminergic systems, much of the methodology is generally applicable for non-human primate fMRI studies and addresses specific problems encountered in imaging awake animals. These are (1) adapting animals to an MRI environment, (2) minimizing head movements, (3) reducing ambient scanning noise levels, and (4) developing multivariate methods of image data analysis suitable for eliciting the dynamic brain response while (5) detecting and deleting outlying observations due to motion artifacts. Procedures are demonstrated for first pre-processing and analyzing responses in a voxel-based approach in a single animal and then proceeding to analyze responses across animals and replicate scans for regions of interest. Collectively, the procedures described provide an approach for fMRI mapping of elicited responses using conventional 1.5T MR scanners.