Biology subjects

Sims, P. A.

Publications and source records attributed to Sims, P. A..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Scalable co-sequencing of RNA and DNA from individual nuclei

The ideal technology for directly investigating the relationship between genotype and phenotype would analyze both RNA and DNA genome-wide and with single-cell resolution. However, existing tools lack the throughput required for comprehensive analysis of complex tumors and tissues. We introduce a highly scalable method for jointly profiling DNA and expression following nucleosome depletion (DEFND-seq). In DEFND-seq, nuclei are nucleosome-depleted, tagmented, and separated into individual droplets for mRNA and genomic DNA barcoding. Once nuclei have been depleted of nucleosomes, subsequent steps can be performed using the widely available 10x Genomics droplet microfluidic technology and commercial kits without experimental modification. We demonstrate the production of high-complexity mRNA and gDNA sequencing libraries from thousands of individual nuclei from both cell lines and archived surgical specimens for associating gene expression phenotypes with both copy number and single nucleotide variants.

genomics↗

bioRxiv2022

Single-Cell Analysis of 5-ALA Intraoperative Labeling Specificity for Glioblastoma

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor, and surgical resection is a key part of the standard-of-care. In fluorescence-guided surgery (FGS), fluorophores are used to differentiate tumor tissue from surrounding normal brain. The heme synthesis pathway converts 5-aminolevulinic acid (5-ALA), a fluorogenic substrate used for FGS, to fluorescent protoporphyrin IX (PpIX). The resulting fluorescence is thought to be specific to transformed glioma cells, but this specificity has not been examined at single-cell level. We performed paired single-cell imaging and RNA sequencing of individual cells (SCOPE-seq2) on human GBM surgical specimens with visible PpIX fluorescence from patients who received 5-ALA prior to surgery. SCOPE-seq2 allows us to simultaneously image PpIX fluorescence and unambiguously identify transformed glioma cells from single-cell RNA-seq (scRNA-seq). We observed that 5-ALA treatment results in labeling that is not specific to transformed tumor cells. In cell culture, we further demonstrated that untransformed cells can be labeled by 5-ALA directly or by PpIX secreted from surrounding transformed cells. In acute slice cultures from mouse glioma models, we showed that 5-ALA preferentially labels GBM tumor tissue over non-neoplastic brain tissue, and that this contrast is not due to blood-brain-barrier disruption. Taken together, our findings support the use of 5-ALA as an indicator of GBM tissue, but not as a specific marker of transformed glioma cells.

cancer biology↗

bioRxiv2022

A cross-disease human microglial framework identifies disease-enriched subsets and tool compounds for microglial polarization.

Human microglia play a pivotal role in neurological diseases, but few targeted therapies that directly modulate microglial state or function exist due to an incomplete understanding of microglial heterogeneity. We use single-cell RNA sequencing to profile live human microglia from autopsies or surgical resections across diverse neurological diseases and central nervous system regions. We observe a central divide between oxidative and heterocyclic metabolism and identify subsets associated with antigen presentation, motility, and proliferation. Specific subsets are enriched in susceptibility genes for neurodegenerative diseases or the disease-associated microglial signature. We validate subtypes in situ with an RNAscope-immunofluorescence pipeline and leverage our dataset as a classification resource, finding that iPSC model systems recapitulate substantial in vivo heterogeneity. Finally, we identify and validate candidates for chemically inducing subtype-specific states in vitro, showing that Camptothecin downregulates the transcriptional signature of disease-enriched subsets and upregulates a signature previously shown to be depleted in Alzheimers.

neuroscience↗

bioRxiv2021

Subcellular and regional localization of mRNA translation in midbrain dopamine neurons

Local translation within excitatory and inhibitory neurons is involved in neuronal development and synaptic plasticity. Despite the extensive dendritic and axonal arborizations of central monoaminergic neurons, the subcellular localization of protein synthesis is not well-characterized in these populations. Here, we investigated mRNA localization and translation in midbrain dopaminergic (mDA) neurons, cells with enormous axonal and dendritic projections, both of which exhibit stimulation-evoked dopamine (DA) release. Using highly-sensitive ribosome-bound RNA-sequencing and imaging approaches in mDA axons, we found no evidence for axonal mRNA localization or translation. In contrast, mDA neuronal dendritic projections into the substantia nigra reticulata (SNr) contain ribosomes and mRNAs encoding the major components of DA synthesis, release, and reuptake machinery. Surprisingly, we also observed dendritic localization of mRNAs encoding synaptic vesicle-related proteins, including those involved in vesicular exocytic fusion. Our results are consistent with a role for local translation in the regulation of DA release from dendrites, but not from axons. Our translatome data further defined a molecular signature of the sparse mDA neurons resident in the SNr, including enrichment of Atp2a3/SERCA3, an ER calcium pump previously undescribed in mDA neurons.

neuroscience↗

bioRxiv2021

Subcellular proteomics of dopamine neurons in the mouse brain reveals axonal enrichment of proteins encoded by Parkinson's disease-linked genes

Dopaminergic neurons modulate neural circuits and behaviors via dopamine release from expansive, long range axonal projections. The elaborate cytoarchitecture of these neurons is embedded within complex brain tissue, making it difficult to access the neuronal proteome using conventional methods. Here, we demonstrate APEX2 proximity labeling within genetically targeted neurons in the mouse brain, enabling subcellular proteomics with cell type-specificity. By combining APEX2 biotinylation with mass spectrometry, we mapped the somatodendritic and axonal proteomes of midbrain dopaminergic neurons. Our dataset reveals the proteomic architecture underlying proteostasis, axonal metabolism, and neurotransmission in these neurons. We find a significant enrichment of proteins encoded by Parkinsons disease-linked genes in striatal dopaminergic axons, including proteins with previously undescribed axonal localization. These proteomic datasets provide a resource for neuronal cell biology, and this approach can be readily adapted for study of other neural cell types.

neuroscience↗

bioRxiv2020

ASCL1 drives induction of a transitory cell state required for repair of the injured neonatal brain

The underlying molecular foundation of neural progenitor diversity and plasticity is critical for understanding repair processes. The neonatal cerebellum with multiple progenitor populations has high regenerative potential. Upon ablation of cerebellar granule cell progenitors at birth, a normally gliogenic Nestin-expressing progenitor (NEP) in the Bergmann glia (Bg) layer (BgL-NEPs) undergoes adaptive reprograming to restore granule neuron production while a white matter NEP (WM-NEPs) reduces interneuron production. However, the cellular states and genes regulating the NEP fate switch are not known. Here using scRNA-seq and fate-mapping, we defined the molecular subtypes of NEPs and their lineages under homeostasis and repair. Five NEP populations comprising two molecular subtypes, Hopx-expressing gliogenic- and Ascl1-expressing neurogenic-NEPs were identified in both states. Furthermore, in the WM, distinct NEP populations generate interneurons or astrocytes, and amongst gliogenic-NEPs, astrocyte and Bg lineages are molecularly separable. Importantly, we uncovered that after injury a new transitory cellular state arises from Hopx-NEPs in the BgL that is defined by initiation of expression of the neurogenic gene Ascl1. Moreover, Ascl1 is required for adaptive reprogramming and the full regenerative capacity of the cerebellum. We thus define new populations of NEPs and identifed the transcription factor responsible for inducing a transitory cell critical for a glial to neural switch in vivo following injury.

developmental biology↗

bioRxiv2020

The functional and phenotypic diversity of single T-cell infiltrates in human colorectal cancer as correlated with clinical outcome

Although degree of T-cell infiltration in CRC was shown to correlate with a positive prognosis, the contribution of phenotypically and functionally distinct T cell subtypes within tumors remains unclear. We analyzed 37,931 single T cells with respect to transcriptome, TCR sequence and 23 cell surface proteins, from tumors and adjacent normal colon of 16 patients. Our comprehensive analysis revealed two phenotypically distinct cytotoxic T cell populations within tumors, including positively prognostic effector memory cells and non-prognostic resident memory cells. These cytotoxic T cell infiltrates transitioned from effector memory to resident memory in a stage-dependent manner. We further defined several Treg subpopulations within tumors. While Tregs overall were associated with positive clinical outcomes, CD38+ peripherally-derived Tregs, phenotypically related to Th17 cells, correlated with poor outcomes independent of cancer stage. Thus, our data highlight the diversity of T cells in CRC and demonstrate the prognostic significance of distinct T cell subtypes, which could inform therapeutic strategies.

immunology↗

bioRxiv2020

Integrating single-cell RNA-seq and imaging with SCOPE-seq2

Live cell imaging allows direct observation and monitoring of phenotypes that are difficult to infer from transcriptomics. However, existing methods for linking microscopy and single-cell RNA-seq (scRNA-seq) have limited scalability. Here, we describe an upgraded version of Single Cell Optical Phenotyping and Expression (SCOPE-seq2) for combining single-cell imaging and expression profiling, with substantial improvements in throughput, molecular capture efficiency, linking accuracy, and compatibility with standard microscopy instrumentation. We introduce improved optically decodable mRNA capture beads and implement a more scalable and simplified optical decoding process. We demonstrate the utility of SCOPE-seq2 for fluorescence, morphological, and expression profiling of individual primary cells from a human glioblastoma (GBM) surgical sample, revealing relationships between simple imaging features and cellular identity, particularly among malignantly transformed tumor cells.Competing Interest StatementZ.L., J.Y. and P.A.S. are listed as inventors on patent applications filed by Columbia University related to the work described here.View Full Text

systems biology↗

bioRxiv2020

Elongation Inhibitors do not Prevent the Release of Puromycylated Nascent Polypeptide Chains from Ribosomes

Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation.

biochemistry↗

bioRxiv2020

Deconvolution of Cell Type-Specific Drug Responses in Human Tumor Tissue with Single-Cell RNA-seq

Precision oncology requires the timely selection of effective drugs for individual patients. An ideal platform would enable rapid screening of cell type-specific drug sensitivities directly in patient tumor tissue and reveal strategies to overcome intratumoral heterogeneity. Here we combine multiplexed drug perturbation in acute slice culture from freshly resected tumors with single-cell RNA sequencing (scRNA-seq) to profile transcriptome-wide drug responses. We applied this approach to glioblastoma (GBM) and demonstrated that acute slice cultures from individual patients recapitulate the cellular and molecular features of the originating tumor tissue. Detailed investigation of etoposide, a topoisomerase poison, and the histone deacetylase (HDAC) inhibitor panobinostat in acute slice cultures revealed cell type-specific responses across multiple patients, including unexpected effects on the immune microenvironment. We anticipate that this approach will facilitate rapid, personalized drug screening to identify effective therapies for solid tumors.

cancer biology↗

bioRxiv2019

High-Throughput Translational Profiling with riboPLATE-seq

Protein synthesis is dysregulated in many diseases, but we lack a systems-level picture of how signaling molecules and RNA binding proteins interact with the translational machinery, largely due to technological limitations. Here we present riboPLATE-seq, a scalable method for generating paired libraries of ribosome-associated and total mRNA. As an extension of the PLATE-seq protocol, riboPLATE-seq utilizes barcoded primers for pooled library preparation, but additionally leverages rRNA immunoprecipitation on whole polysomes to measure ribosome association (RA). We compare RA to its analogue in ribosome profiling and RNA sequencing, translation efficiency, and demonstrate both the performance of riboPLATE-seq and its utility in detecting translational alterations induced by specific inhibitors of protein kinases.

systems biology↗

bioRxiv2019

Single-cell profiling and SCOPE-seq reveal the lineage dynamics of adult neurogenesis and NOTUM as a key V-SVZ regulator

Neural stem cells (NSCs) and their progeny reside in specialized niches in the adult mammalian brain where they generate new neurons and glia throughout life. Adult NSCs of the ventricular-subventricular zone (V-SVZ) are prone to rapid exhaustion; thus timely, context-dependent neurogenesis demands adaptive signaling among the vast number of neighboring progenitors nestled between the ventricular surface and nearby blood vessels. To dissect adult neuronal lineage progression and regulation, we profiled >56,000 V-SVZ and olfactory bulb (OB) cells by single-cell RNA-sequencing (scRNA-seq). Our analyses revealed the diversity of V-SVZ-derived OB neurons, the temporal dynamics of lineage progression, and a key intermediate NSC population enriched for expression of Notum, which encodes a secreted WNT antagonist. Single Cell Optical Phenotyping and Expression (SCOPE-seq), a technology linking live cell imaging with scRNA-seq, uncovered dynamic control of cell size concomitant with NSC differentiation with Notum+ NSCs at a critical size poised for cell division, and a preference of NOTUM surface binding to neuronal precursors with active WNT signaling. Finally, in vivo pharmacological inhibition of NOTUM significantly expanded neuronal precursor pools in the V-SVZ. Our findings highlight a critical regulatory state during NSC activation marked by NOTUM, a secreted enzyme that ensures efficient neurogenesis by preventing WNT signaling activation in NSC progeny.

neuroscience↗

bioRxiv2019

Single-cell based elucidation of molecularly-distinct glioblastoma states and drug sensitivity

Glioblastoma heterogeneity and plasticity remain controversial, with proposed subtypes representing the average of highly heterogeneous admixtures of independent transcriptional states. Single-cell, protein-activity-based analysis allowed full quantification of >6,000 regulatory and signaling proteins, thus providing a previously unattainable single-cell characterization level. This helped identify four novel, molecularly distinct subtypes that successfully harmonize across multiple GBM datasets, including previously published bulk and single-cell profiles and single cell profiles from seven orthotopic PDX models, representative of prior subtype diversity. GBM is thus characterized by the plastic coexistence of single cells in two mutually-exclusive developmental lineages, with additional stratification provided by their proliferative potential. Consistently, all previous subtypes could be recapitulated by single-cell mixtures drawn from newly identified states. Critically, drug sensitivity was predicted and validated as highly state-dependent, both in single-cell assays from patient-derived explants and in PDX models, suggesting that successful treatment requires combinations of multiple drugs targeting these distinct tumor states.\n\nSignificanceWe propose a new, 4-subtype GBM classification, which harmonizes across bulk and single-cell datasets. Single-cell mixtures from these subtypes effectively recapitulate all prior classifications, suggesting that the latter are a byproduct of GBM heterogeneity. Finally, we predict single-cell level activity of three clinically-relevant drugs, and validate them in patient-derived explant.

systems biology↗

bioRxiv2019

A single-cell reference map for human blood and tissue T cell activation reveals functional states in health and disease

Human T cells coordinate adaptive immunity by localization in diverse tissue sites, though blood T cells are the most readily studied. Here, we used single-cell RNA-seq to define the functional responses of T cells isolated from human lungs, lymph nodes, bone marrow, and blood to TCR-stimulation. We reveal how human T cells in tissues relate to those in blood, and define activation states for CD4+ and CD8+T cells across all sites, including an interferon-response state for CD4+T cells and distinct effector states for CD8+T cells. We further show how profiles of individual tumor-associated T cells can be projected onto this healthy reference map, revealing their functional state.

immunology↗

bioRxiv2018

De novo Gene Signature Identification from Single-Cell RNA-Seq with Hierarchical Poisson Factorization

Common approaches to gene signature discovery in single cell RNA-sequencing (scRNA-seq) depend upon predefined structures like clusters or pseudo-temporal order, require prior normalization, or do not account for the sparsity of single cell data. We present single cell Hierarchical Poisson Factorization (scHPF), a Bayesian factorization method that adapts Hierarchical Poisson Factorization [1] for de novo discovery of both continuous and discrete expression patterns from scRNA-seq. scHPF does not require prior normalization and captures statistical properties of single cell data better than other methods in benchmark datasets. Applied to scRNA-seq of the core and margin of a high-grade glioma, scHPF uncovers marked differences in the abundance of glioma subpopulations across tumor regions and subtle, regionally-associated expression biases within glioma subpopulations. scHFP revealed an expression signature that was spatially biased towards the glioma-infiltrated margins and associated with inferior survival in glioblastoma.

systems biology↗

bioRxiv2018

An Optically Decodable Bead Array for Linking Imaging and Sequencing with Single-Cell Resolution

Optically decodable beads link the identity of an analyte or sample to a measurement through an optical barcode, enabling libraries of biomolecules to be captured on beads in solution and decoded by fluorescence. This approach has been foundational to microarray, sequencing, and flow-based expression profiling technologies. We have combined microfluidics with optically decodable beads to link phenotypic analysis of living cells to sequencing. As a proof-of-concept, we applied this to demonstrate an accurate and scalable tool for connecting live cell imaging to single-cell RNA-Seq called Single Cell Optical Phenotyping and Expression (SCOPE-Seq).

systems biology↗

bioRxiv2018

Widespread Alterations in Translation Elongation in the Brain of Juvenile Fmr1 Knock-Out Mice

FMRP is a polysome-associated RNA-binding protein encoded by Fmr1 that is lost in Fragile X syndrome. Increasing evidence suggests that FMRP regulates both translation initiation and elongation, but the gene-specificity of these effects is unclear. To elucidate the impact of Fmr1 loss on translation, we used ribosome profiling for genome-wide measurements of ribosomal occupancy and positioning in the cortex of 24 day-old Fmr1 knock-out mice. We found a remarkably coherent reduction in ribosome footprint abundance per mRNA for previously identified, high-affinity mRNA binding partners of FMRP, and an increase for terminal oligo-pyrimidine (TOP) motif-containing genes canonically controlled by mTOR-4EBP-eIF4E signaling. Amino acid motif- and gene-level analyses both showed a widespread reduction of translational pausing in Fmr1 knock-out mice. Our findings are consistent with a model of FMRP-mediated regulation of both translation initiation through eIF4E and elongation that is disrupted in Fragile X syndrome.

molecular biology↗

bioRxiv2018

Quantitative Assessment of Protein Activity in Orphan Tissues and Single Cells Using the metaVIPER Algorithm

We and others have shown that transition and maintenance of biological states is controlled by master regulator protein, which can be inferred by interrogating tissue-specific regulatory models (interactomes) with transcriptional signatures, using the VIPER algorithm. Yet, some tissues may lack molecular profiles necessary for interactome inference (orphan tissues), or, as for single cells isolated from heterogeneous samples, their tissue context may be undetermined. To address this problem, we introduce metaVIPER, a novel algorithm designed to assess protein activity in tissue-independent by integrative analysis of multiple, non-tissue-matched interactomes. This assumes that transcriptional targets of each protein will be recapitulated by one or more available interactome. We confirmed the algorithms value in assessing protein dysregulation induced by somatic mutations, as well as in assessing protein activity in orphan tissues and, most critically, in single cells, thus allowing transformation of noisy and potentially biased RNA-Seq signatures into reproducible protein-activity signatures.

systems biology↗