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Fan, J.

Publications and source records attributed to Fan, J..

At least 55 records · Page 3Linked to original sources

Brain-wide Genome Editing via STEP-RNPs for Treatment of Angelman Syndrome

Brain-wide genome-editing remains a major hurdle for the treatment of neurogenetic disorders. Here, we report a non-viral, non-nanoparticle, chemical modification-based method, called Stimuli-Responsive Traceless Engineering Platform (STEP) that achieves highly efficient and brain-wide genome-editing of neurons in mice. Using cholesterol-based STEP as a lead, we show that a single administration of STEP-ribonucleoproteins (RNPs) results in functional genetic rescue with significant improvements across a battery of neurobehavioral domains in the Angelman syndrome (AS) mouse model. No significant off-target events or general toxicity effects are observed. STEP-RNPs are also highly efficient at editing human neurons and cortical brain organoids differentiated from AS patient-derived iPSCs. scRNA-seq analysis confirms functional genetic rescue via reactivation of Ube3a/UBE3A expression in human and mouse STEP-RNP-treated neuronal cells. Genome editing via STEP-RNPs has broad applications and the potential to treat many other neurogenetic disorders.

genetics↗

Modulation of Neutrophil Function by Foetal Bovine Serum and Type I Collagen

Immune regulation plays a crucial role during the regeneration process, and it determines the fate of inflammation after tissue injury or infection. Neutrophils serve as the primary immune cells recruited to the site of inflammation, initiating and directing the subsequent inflammatory cascade following implantation. As neutrophils are highly sensitive to environmental cues, commonly used culture supplements and molecules may themselves influence neutrophil function and consequently affect the interpretation of biomaterial-induced immune responses. This study examined how foetal bovine serum (FBS), a standard in vitro culture supplement, affects human peripheral blood neutrophils when supplied either in the culture medium or as a surface coating on 3D-printed polycaprolactone (PCL) scaffolds. The response to type I collagen coating was also assessed as a biologically relevant comparator. Neutrophil activity was evaluated by assessing metabolic activity and metabolomic profiles, reactive oxygen species (ROS) production, and inflammation-related markers via a high-throughput proximity extension assay. Type I collagen coating modified the metabolomic profile of neutrophils and MMP-9 release but had minimal effect on ROS generation. In contrast, the presence of FBS in the culture medium significantly influenced neutrophil behavior, leading to significant changes in metabolic activity, cytotoxicity, and the secretion of inflammation-associated molecules, even at concentrations as low as 1% (v/v). These findings highlight the importance of assessing the use of FBS in neutrophil culture models, particularly those isolated from humans, and emphasize the need to develop alternative platforms for investigating neutrophil-biomaterial interactions in a more physiologically relevant manner.

immunology↗

RainBar: Optical Barcoding for Pooled Live-Cell Imaging with Single-Cell Resolution

High-throughput pooled screening has advanced functional genomics, but most existing methods rely on endpoint sequencing and are blind to dynamic, time-resolved phenotypes. We developed RainBar (Rainbow Barcodes), an optical barcoding system that supports pooled live-cell imaging with single-cell resolution. RainBar uses lentiviral co-delivery of spectrally distinct nuclear and cytoplasmic fluorescent proteins to encode up to 64 unique perturbations per well. To mitigate barcode recombination and improve decoding accuracy, we employed single-template viral production, codon diversification, and a ratio-based spectral unmixing pipeline tailored to overlapping fluorophores. An inverted cytoplasmic segmentation approach and multilayer perceptron classifier enabled accurate barcode identification in both arrayed and pooled formats. As a proof of concept, we applied RainBar to dissect NF-{kappa}B signaling dynamics in epithelial cells. Live imaging of RelA translocation uncovered stimulus-specific kinetics: IL-1{beta} triggered rapid recovery, while TNF induced prolonged nuclear localization. In pooled CRISPRi screens, RainBar recovered known NF-{kappa}B regulators (e.g., IL1R1, MYD88, TNFRSF1A) and highlighted additional modulators, including the Ino80 chromatin remodeling complex subunits and KAT2A acetyltransferase. Together, these results position RainBar as a flexible platform for multiplexed, image-based functional genomics, with potential to reveal dynamic signaling architectures across diverse cellular contexts in live cells.

genomics↗

Machine Vision Enabled Lateral Flow Immunoassay Using Functionalized Gold Nanoparticles for Point-of-Care Cardiac Biomarker Detection

Acute myocardial infarction (AMI) remains one of the most prevalent and fatal cardiovascular disease. Given the critical diagnostic significance of cardiac troponin I (cTnI) and myoglobin (Myo) in AMI, there is an urgent clinical demand for rapid and accurate detection methods to improve patient outcomes and reduce mortality. To meet this need, we developed a sensing platform that integrates functionalized gold nanoparticle-based lateral flow immunoassay (AuNPs-LFIA) with a machine vision model, enabling rapid quantitative detection of cTnI and Myo. By covalently conjugating antibodies to gold nanoparticles (AuNPs@Ab), we achieved greater probe specificity and stability. The integration of machine vision algorithms allowed quantitative readouts within 8 minutes, a 46.7% improvement of the detection time compared to conventional methods (15 minutes). The platform achieved limits of detection of 0.224 ng/mL for Myo and 0.071 ng/mL for cTnI, with excellent correlation to commercial kits (R2 > 0.99). Overall, these results demonstrate that machine vision-enhanced AuNPs-LFIA offers an efficient, sensitive and reliable strategy for point-of- care testing (POCT) in cardiovascular diagnostics, particularly in resource-limited settings.

bioengineering↗

CellBin:a generalist framework to process spatial omics data to cell level

Spatial omics has rapidly expanded with increasingly diverse imaging modalities, molecular targets, and chip sizes. However, no general framework currently exists to construct cell level matrices that are robust across platforms and omics types. Here we present CellBin, a universal and scalable frame-work that unifies image stitching, cell segmentation, and spot-to-cell mapping for multiple spatial omics technologies. CellBin integrates a multi-field weighted stitching algorithm for large-area images, a family of U-Net-based models trained across diverse staining modalities, and an optimized computational architecture for high-throughput processing. Across five technological platforms and three omics data types, CellBin achieves robust segmentation and accurate single-cell matrix construction, consistently outperforming seven state-of-the-art methods in F1-score, cell size precision, and annotation accuracy. By providing a generalizable, cross-platform solution, CellBin bridges multiple spatial omics, enabling unified, high-resolution cell level analyses across technologies.

bioinformatics↗

Multi-site Assessment of Methods for Cell Preservation Upstream of Single Cell RNA Sequencing

Single cell RNA sequencing (scRNA-seq) is a revolutionary technique to identify cell types and their molecular phenotype in heterogeneous biological specimens. ScRNA-seq typically requires fresh, high quality single cell suspensions that are processed immediately to preserve their molecular profiles. This presents a challenge for samples with long preparation times and prevents collection at remote sites lacking the required instrumentation for sample processing. Recently, several commercial assays have been released that enable sample preservation at the time of collection either via fixation or cryopreservation, allowing for sample processing to occur months after the initial collection. The Association of Biomolecular Research Facilities (ABRF) DNA Sequencing (DSRG) and Genomics Bioinformatics (GBiRG) Research Groups have undertaken a cross-platform, multi-site study to assess the performance and reproducibility of three platforms: a) 10x Genomics FLEX, b) Parse Bioscience Evercode WT v2 and c) Honeycomb Bio HIVE. Total leukocytes were isolated from a single healthy individual using the EasySep RBC depletion reagent. Cells were then characterized by collecting a 21-color flow cytometry dataset for reference and the remaining material was used for scRNA-seq procedures where different sites then processed either the fixed or cryopreserved cells for each method. We evaluated performance of each method across traditional scRNA-seq quality control metrics and analysis applications, including gene/transcript detection sensitivity, cell type discovery and annotation, and differential expression. We demonstrate that data from the methods tested can be effectively integrated and produce concordant results with regard to cell type annotation and relative abundance, though we observe platform-specific differences in the expression of a subset of genes. Preservation-based methods also show better retention of fragile granulocyte populations compared with fresh samples processed using the 10x 3 workflow. The improvements to preservation methods are changing the way research is conducted and our thorough investigation into the performance of each method provides a valuable resource to help scientists determine the most appropriate single cell preservation workflow given their sample collection logistics and laboratory infrastructure constraints.

genomics↗

MgAl-layered double hydroxide nanoparticles mediated oral delivery of dsRNA silencing jhp and chi genes with sublethal effects in Phthorimaea absoluta

BACKGROUNDThe use of RNA interference (RNAi)-based technology for the management of agricultural insect pests is gaining momentum worldwide. A prerequisite for successful control by RNAi is the formulation of a highly efficient and stable double-stranded RNA (dsRNA). Here, we investigated the use of magnesium aluminum-layered double hydroxide (MgAl-LDH or LDH) nanoparticles complexed with dsRNA, termed BioClayTM, to target the juvenile hormone inducible protein (jhp) and the chitin synthase A (chi) genes of the South American tomato pinworm Phthorimaea (=Tuta) absoluta, a key pest of tomato crop. RESULTSDegradation assays showed that higher amounts of RNase A degraded dsRNA complexed with the LDH nanoparticles while lower RNase A amounts did not degrade the dsRNA. Furthermore, our findings revealed that dsRNA is stable after 60 min of incubation when in buffer solutions at pH 5 and pH 9, while naked dsRNA showed slight degradation at pH 11. Also, BioClayTM and naked dsRNAs targeting jhp and chi genes did not significantly increase larval mortality in P. absoluta, but induced sublethal phenotypes through reduced pupal weight and adult emergence. Gene expression analysis revealed significant reduction of 73% and 48% in transcript abundance of chi gene in larvae that fed on chi-dsRNA and BioClayTM chi-dsRNA after 72 h, respectively, while a 39% reduction in transcript of jhp gene was recorded only in larvae fed on BioClayTM jhp-dsRNA. CONCLUSIONOur findings provide critical insights into the significant impact of the use of dsRNA technology for targeted gene disruptions affecting important traits in P. absoluta and highlight the potential and opportunities that remain to be explored in the formulation and successful delivery of dsRNA-based active substances in pest management. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/683103v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@b4574forg.highwire.dtl.DTLVardef@1d0113borg.highwire.dtl.DTLVardef@11b5416org.highwire.dtl.DTLVardef@12da956_HPS_FORMAT_FIGEXP M_FIG C_FIG MgAl-LDH nanoparticles used as carrier to deliver dsRNA-based active substances hold potential to target jhp and chi candidate genes in Phthorimaea absoluta for the sustainable management of the pest.

bioengineering↗

Exploring endothelial cell environments across organs in spatially resolved omics data

Endothelial cells are ubiquitously present in the human body and line the luminal surface of blood and lymphatic vessels. The oxygen-dependence of cells impacts their proximity to blood vessels, and consequently, to endothelial cells depending on their functional properties and priorities. This paper presents cell-to-nearest-endothelial-cell distance distributions for various cell types using 399 spatially resolved omics datasets from 14 studies comprising 12 tissue types with a total of 47,349,496 cells. Additionally, we developed an open-source web-based interactive tool, Cell Distance Explorer, that allows researchers to interactively visualize cell graphs and linkages in 2D and 3D datasets. Finally, we present a hierarchical neighborhood analysis focused on the endothelial cell neighborhoods in small and large intestine datasets. This paper provides an open-access resource (datasets, tools, and analyses) to characterize and compare cell distances and cell neighborhoods in spatially resolved omics data.

bioinformatics↗

EWS::WT1 Isoform-Dependent Regulation of Neogenes in Desmoplastic Small Round Cell Tumors

Desmoplastic small round cell tumor (DSRCT) is a rare, aggressive sarcoma characterized by the pathognomonic EWS::WT1 fusion protein (FP), an oncogenic chimeric transcription factor (OCTF) resulting from the t(11;22)(p13;q12) translocation. Recent studies have identified "neogenes" (NGs), genes normally silent in normal tissues but transcriptionally activated by OCTFs, as potential tumor-specific markers in fusion-driven cancers. In this study, we investigated the expression and regulation of DSRCT-specific NGs (DSRCT_NGs) using multimodal data across different cohorts of patients, PDX, and cell line data. We evaluated bulk and single-nucleus RNA sequencing of patient specimens from MD Anderson Cancer Center, revealing the robust ability for DSRCT_NGs to distinguish FP-positive DSRCT from samples failing detection of the EWS::WT1 FP. To elucidate the regulatory role of the EWS::WT1 FP in driving NG expression, we performed knockdown experiments in four DSRCT cell lines. This consistently resulted in a reduction of DSRCT_NG expression. Isoform-specific expression of EWS::WT1 in LP9 and MeT-5A mesothelial cells revealed that the E-KTS isoform of EWS::WT1 predominantly drives DSRCT_NG expression. Mechanistically, ATAC-seq and ChIP-seq analyses demonstrated that EWS::WT1 directly binds to accessible chromatin regions near NG transcription start sites, enriched for WT1 motifs and active histone marks. Integration of Hi-ChIP data further revealed that EWS::WT1 facilitates long-range enhancer-promoter looping at DSRCT_NG loci, promoting the expression of nearby genes. Collectively, these findings establish DSRCT_NGs as direct transcriptional outputs of the EWS::WT1 FP and implicate their loci as regulatory regions of the DSRCT transcriptome. Their fusion-dependent expression, chromatin accessibility, and promoter-enhancer connectivity underscore their potential utility as highly specific biomarkers and therapeutic targets in DSRCT.

genomics↗

Modulating Glutamine Metabolism Reprograms Pro-Inflammatory Differentiation in Macrophages

Previous in vivo studies demonstrated that JHU083/DON, a glutamine analog drug, potently reprograms M1/M2 macrophages. To determine whether these effects are direct or indirect, we utilized an in vitro murine bone marrow-derived macrophage (BMDM) model, which recapitulates macrophage differentiation and polarization processes, to examine the impact of DON on the M1 macrophages. DON was applied during M1 differentiation or to fully polarized M1 macrophages, revealing that glutamine inhibition initially suppressed M1 activity but later enhanced it, resulting in sustained pro-inflammatory activation. Multi-omics analyses (bulk RNA-seq and LC-MS), time-course assays, and glutamine depletion experiments consistently suggested that prolonged glutamine inhibition elevates glutamine levels, which sustain pro-inflammatory gene transcription. In contrast, M2 and tumor-associated macrophages (TAM), which are immunosuppressive, were more susceptible to DON, leading to functional suppression. Collectively, our findings uncover stage-specific mechanisms by which glutamine inhibition modulates M1 polarization, offering a mechanistic rationale for therapeutic strategies that sustain pro-inflammatory, anti-tumor macrophage activity while concurrently suppressing immunosuppressive myeloid subsets in cancer.

cancer biology↗

Impact of Data Quality on Deep Learning Prediction of Spatial Transcriptomics from Histology Images

Spatial transcriptomic technologies enable high-throughput quantification of gene expression at specific locations across tissue sections, facilitating insights into the spatial organization of biological processes. However, high costs associated with these technologies have motivated the development of deep learning methods to predict spatial gene expression from inexpensive hematoxylin and eosin-stained histology images. While most efforts have focused on modifying model architectures to boost predictive performance, the influence of training data quality remains largely unexplored. Here, we investigate how variation in molecular and image data quality stemming from differences in spatial transcriptomic technologies impact deep learning-based gene expression prediction from histology images. To identify the aspects of data quality that impact predictive performance, we conducted in silico ablation experiments, which showed that increased sparsity and noise in molecular data degraded predictive performance, while in silico rescue experiments via imputation provided only limited improvements that failed to generalize beyond the test set. Likewise, reduced image resolution can degrade predictive performance and further impacts model interpretability. We further demonstrate that these data quality-driven effects are reproducible across multiple spatial transcriptomics datasets and remain consistent when using alternative feature extractors and model architectures. Overall, our results show how improving data quality provides an orthogonal strategy to tuning model architecture in spatial transcriptomics-based predictive modeling, highlighting the need to account for technology-specific limitations that directly impact data quality when developing predictive methodologies.

bioinformatics↗

Nanopore-Based Glycan Sequencing via Fragmentation-Reassembly Strategy

Glycan sequencing remains a major analytical challenge due to the high structural complexity, extensive branching, and isomeric diversity of glycans. Nanopore sensing has emerged as a promising single-molecule platform for glycan analysis, but existing strategies--such as hydrolysis and strand sequencing, face significant limitations when applied to highly branched or non-linear glycans. Here we report the first experimental realization of glycan assembly sequencing, a nanopore-based strategy that reconstructs full glycan structures from individually analyzed fragments. Using a biantennary complex-type N-glycan as a model, we performed controlled enzymatic digestion to generate structurally defined oligosaccharide fragments. An engineered -HL nanopore was developed for enhanced glycan recognition, enabling the construction of a multidimensional electrical fingerprinting library. Fragment signals from unknown samples were matched to this reference dataset, and full-sequence reconstruction was achieved via fragment reassembly based on set-theoretic integration of structural candidates. This study establishes glycan assembly sequencing as a viable and modular approach to resolve complex glycoforms using nanopore technologies. The method expands the accessible sequence space beyond what is achievable with conventional strategies and provides a foundation for scalable, high-resolution glycomic analysis.

bioengineering↗

Distributed control circuits across a brain-and-cord connectome

Just as genomes revolutionized molecular genetics, connectomes (maps of neurons and synapses) are transforming neuroscience. To date, the only species with complete connectomes are worms1-3 and sea squirts4 (103-104 synapses). By contrast, the fruit fly is more complex (108 synaptic connections), with a brain that supports learning and spatial memory5,6 and an intricate ventral nerve cord analogous to the vertebrate spinal cord7-11. Here we report the first densely reconstructed adult fly connectome that unites the brain and ventral nerve cord, and we leverage this resource to investigate principles of neural control. We show that effector neurons (motor neurons, endocrine cells and efferent neurons targeting the viscera) are primarily influenced by sensory neurons in the same body part, forming local feedback loops. These local loops are linked by long-range circuits involving ascending and descending neurons organized into behavior-centric modules. Single ascending and descending neurons are often positioned to influence the voluntary movements of multiple body parts, together with the endocrine cells or visceral organs that support those movements. Brain regions involved in learning and navigation supervise these circuits. These results reveal an architecture that is distributed, parallelized and embodied, reminiscent of distributed control architectures in engineered systems12,13.

neuroscience↗

Expanding the DNA Motif Lexicon of the Transcriptional Regulatory Code

Transcriptional regulatory sequences in metazoans contain intricate combinations of transcription factor (TF) motifs. Stereospecific arrangements of simple motifs constitute composite elements (CEs) that enhance DNA-protein interaction specificity and enable combinatorial regulatory logic. Despite their importance, CEs remain underexplored. We advance CE discovery and functional characterization by developing an integrated framework that combines computational prediction, experimental testing and deep learning. The extended TF motif catalog comprises both synergistic and counteracting CEs, which are supported by evidence of TF binding in vivo and in vitro. A deep learning model GRACE trained on customized massively parallel reporter assays learns the lexicon of CEs at single-nucleotide resolution. Comparative analysis with a neural network model trained on chromatin accessibility demonstrates striking convergence and distinctions within the expanded regulatory lexicon, enabling joint predictions of motif contributions and the impact of variants on chromatin structure and transcriptional activity in diverse cellular contexts.

genomics↗

Repetitive mild traumatic brain injury causes neuronal damage in the APP/PS1 mouse model of Alzheimer's disease without an enduring impact on amyloid pathology, sleep, or epileptiform activity

Traumatic Brain Injury (TBI) is a known risk factor for Alzheimers disease and related neurodegenerative diseases. Sleep disturbances and epileptiform abnormalities can appear after TBI and may contribute to the development of neuropathology. In this study, we characterized sleep, epileptiform activity, and neuropathology after repetitive mild traumatic brain injury (rmTBI) in a mouse model of Alzheimers disease. We used the Closed Head Impact Model of Engineered Rotational Acceleration (CHIMERA) to deliver rmTBI or sham (control) treatment to 6-month-old APP/PS1 mice (N=19). One month post-injury, we implanted electroencephalogram (EEG) and electromyographic (EMG) electrodes, recorded for 72 hours, and then collected brain tissue and blood plasma. Our assessment of sleep architecture showed that time spent in vigilance state was not affected by the rmTBI one month post-injury; however, power spectra analysis showed a shift towards higher frequencies in the rmTBI group during non-rapid eye movement (NREM) sleep. Epileptiform activity did not differ between sham and rmTBI. Compared to sham controls, the rmTBI group showed higher neurofilament light (NF-L), but not glial-fibrillary acidic protein (GFAP) in blood plasma and no change in A{beta} pathology. These results indicate sustained neurological injury in the APP/PS1 mice one month after rmTBI without affecting amyloid deposition in the brain. Our study suggests that rmTBI can induce neural injury without causing enduring sleep disruption, seizures, and exacerbation of amyloidosis in the APP/PS1 mouse model.

neuroscience↗

Spatiotemporal transcriptomic analysis during cold ischemic injury to the murine kidney reveals compartment-specific changes

BackgroundKidney transplantation is the preferred treatment strategy for end-stage kidney disease. Deceased donor kidneys usually undergo cold storage until kidney transplantation, leading to cold ischemia injury that may contribute to poor graft outcomes. However, the molecular characterization of potential mechanisms of cold ischemia injury remains incomplete. ResultsTo bridge this knowledge gap, we leveraged the 10x Visium spatial transcriptomic technology to perform full transcriptome profiling of murine kidneys subject to varying durations of cold ischemia typical in a deceased donor kidney transplant setting. We developed a computational workflow to identify and compare spatiotemporal transcriptomic changes that accompany the injury pathophysiology in a tissue compartment-specific manner. We identified proportional enrichment of oxidative phosphorylation (OXPHOS) genes with increasing duration of cold ischemia injury within the oxygen-lean inner medulla region, suggestive of atypical metabolic presentation. This was distinct in cold ischemia injury tissue compared to warm ischemia-reperfusion kidney injury tissue. Spatiotemporal trends were validated by qPCR and immunofluorescence in a larger cohort of mice. We provide an interactive online browser at https://jef.works/CellCarto-ColdIschemia/ to facilitate exploration of our results by the broader scientific and clinical community. ConclusionsAltogether, our spatiotemporal transcriptomic analysis identified coordinated molecular changes within metabolic pathways such as OXPHOS deep within the cold ischemic kidney, highlighting the need for increased attention to the inner medulla and potential opportunities for new insights beyond those available from superficial biopsy-focused tissue examinations.

bioinformatics↗

Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

Immune checkpoint inhibitor (ICB) therapy for many cancers remains limited in patients overall response rate. Discovery and development of more effective combinatorial approaches is urgent. Here, through CRISPR/Cas9 genetic screens, we identify DOT1L as a versatile epigenetic factor that functions to suppress tumor-intrinsic immunity through a dual mechanism. Depletion of DOT1L induces the expression of transposable elements and subsequent type I interferon (IFN) response, and meanwhile lowers ZEB1 levels to further unleash the expression of immune-related genes. In turn, we demonstrate that DOT1L loss or treatment with the clinical stage inhibitor EPZ-5676 sensitizes tumors to ICB with increased immune infiltration in mice. More importantly, EPZ-5676 treatment alone is sufficient to enhance antitumor immunity in humanized mice. TCGA data analysis reveals an inverse correlation between DOT1L expression and IFN signatures across multiple cancer types. These findings provide a rationale for targeting DOT1L to improve tumor immunogenicity and overcome immunotherapy resistance. One Sentence SummaryCRISPR genetic screens identified DOT1L as a potential suppressor of tumor intrinsic immunogenicity

cell biology↗

The China Brain Multi-omics Atlas Project (CBMAP)

The China Brain Multi-omics Atlas Project (CBMAP) aims to generate a comprehensive molecular reference map of over 1,000 human brains (Phase I), spanning a broad age range and multiple regions in China, to address the underrepresentation of East Asian populations in brain research. By integrating genome, epigenome, transcriptome, proteome (including multiple post-translational modifications), and metabolome data, CBMAP is set to provide a rich and invaluable resource for investigating the molecular underpinnings of aging-related brain phenotypes and neuropsychiatric disorders. Leveraging high-throughput omics data and advanced technologies, such as spatial transcriptomics, proteomics, and single-nucleus 3D chromatin structure analysis, this atlas will serve as a crucial resource for the brain science community, illuminating disease mechanisms and enhancing the utility of data from genome-wide association studies (GWAS). CBMAP is also poised to accelerate drug discovery and precision medicine for brain disorders.

neuroscience↗