Publications by authors named "Stephen R Quake"

263 Publications

Single cell biology-a Keystone Symposia report.

Ann N Y Acad Sci 2021 Oct 3. Epub 2021 Oct 3.

Department of Biochemistry and BioFrontiers Institute, University of Colorado Boulder, Boulder, Colorado.

Single cell biology has the potential to elucidate many critical biological processes and diseases, from development and regeneration to cancer. Single cell analyses are uncovering the molecular diversity of cells, revealing a clearer picture of the variation among and between different cell types. New techniques are beginning to unravel how differences in cell state-transcriptional, epigenetic, and other characteristics-can lead to different cell fates among genetically identical cells, which underlies complex processes such as embryonic development, drug resistance, response to injury, and cellular reprogramming. Single cell technologies also pose significant challenges relating to processing and analyzing vast amounts of data collected. To realize the potential of single cell technologies, new computational approaches are needed. On March 17-19, 2021, experts in single cell biology met virtually for the Keystone eSymposium "Single Cell Biology" to discuss advances both in single cell applications and technologies.
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http://dx.doi.org/10.1111/nyas.14692DOI Listing
October 2021

RNA splicing programs define tissue compartments and cell types at single cell resolution.

Elife 2021 Sep 13;10. Epub 2021 Sep 13.

Department of Biochemistry, Department of Biomedical Data Science, Stanford University, Stanford, CA, United States.

The extent splicing is regulated at single-cell resolution has remained controversial due to both available data and methods to interpret it. We apply the SpliZ, a new statistical approach, to detect cell-type-specific splicing in >110K cells from 12 human tissues. Using 10x data for discovery, 9.1% of genes with computable SpliZ scores are cell-type-specifically spliced, including ubiquitously expressed genes and . These results are validated with RNA FISH, single-cell PCR, and Smart-seq2. SpliZ analysis reveals 170 genes with regulated splicing during human spermatogenesis, including examples conserved in mouse and mouse lemur. The SpliZ allows model-based identification of subpopulations indistinguishable based on gene expression, illustrated by subpopulation-specific splicing of classical monocytes involving an ultraconserved exon in . Together, this analysis of differential splicing across multiple organs establishes that splicing is regulated cell-type-specifically.
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http://dx.doi.org/10.7554/eLife.70692DOI Listing
September 2021

Quantifying rapid bacterial evolution and transmission within the mouse intestine.

Cell Host Microbe 2021 09 1;29(9):1454-1468.e4. Epub 2021 Sep 1.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA. Electronic address:

Due to limitations on high-resolution strain tracking, selection dynamics during gut microbiota colonization and transmission between hosts remain mostly mysterious. Here, we introduced hundreds of barcoded Escherichia coli strains into germ-free mice and quantified strain-level dynamics and metagenomic changes. Mutations in genes involved in motility and metabolite utilization are reproducibly selected within days. Even with rapid selection, coprophagy enforced similar barcode distributions across co-housed mice. Whole-genome sequencing of hundreds of isolates revealed linked alleles that demonstrate between-host transmission. A population-genetics model predicts substantial fitness advantages for certain mutants and that migration accounted for ∼10% of the resident microbiota each day. Treatment with ciprofloxacin suggests interplay between selection and transmission. While initial colonization was mostly uniform, in two mice a bottleneck reduced diversity and selected for ciprofloxacin resistance in the absence of drug. These findings highlight the interplay between environmental transmission and rapid, deterministic selection during evolution of the intestinal microbiota.
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http://dx.doi.org/10.1016/j.chom.2021.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8445907PMC
September 2021

The cell as a bag of RNA.

Authors:
Stephen R Quake

Trends Genet 2021 Aug 27. Epub 2021 Aug 27.

Stanford University and Chan Zuckerberg Biohub, 318 Campus Drive, Stanford, CA 94305, USA. Electronic address:

Genomic sequencing has provided insight into the genetic characterization of many organisms, and we are now seeing sequencing technologies turned towards phenotypic characterization of cells, tissues, and whole organisms. In particular, single-cell transcriptomic techniques are revolutionizing certain aspects of cell biology and enabling fundamental discoveries about cellular diversity, cell state, and cell type identity. I argue here that much of this progress depends on abstracting one's view of the cell to regard it as a 'bag of RNA'.
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http://dx.doi.org/10.1016/j.tig.2021.08.003DOI Listing
August 2021

Gut cytokines modulate olfaction through metabolic reprogramming of glia.

Nature 2021 08 21;596(7870):97-102. Epub 2021 Jul 21.

Immunology Discovery, Genentech, Inc., South San Francisco, CA, USA.

Infection-induced aversion against enteropathogens is a conserved sickness behaviour that can promote host survival. The aetiology of this behaviour remains poorly understood, but studies in Drosophila have linked olfactory and gustatory perception to avoidance behaviours against toxic microorganisms. Whether and how enteric infections directly influence sensory perception to induce or modulate such behaviours remains unknown. Here we show that enteropathogen infection in Drosophila can modulate olfaction through metabolic reprogramming of ensheathing glia of the antennal lobe. Infection-induced unpaired cytokine expression in the intestine activates JAK-STAT signalling in ensheathing glia, inducing the expression of glial monocarboxylate transporters and the apolipoprotein glial lazarillo (GLaz), and affecting metabolic coupling of glia and neurons at the antennal lobe. This modulates olfactory discrimination, promotes the avoidance of bacteria-laced food and increases fly survival. Although transient in young flies, gut-induced metabolic reprogramming of ensheathing glia becomes constitutive in old flies owing to age-related intestinal inflammation, which contributes to an age-related decline in olfactory discrimination. Our findings identify adaptive glial metabolic reprogramming by gut-derived cytokines as a mechanism that causes lasting changes in a sensory system in ageing flies.
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http://dx.doi.org/10.1038/s41586-021-03756-0DOI Listing
August 2021

Reciprocal repulsions instruct the precise assembly of parallel hippocampal networks.

Science 2021 06;372(6546):1068-1073

Department of Biology, Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA.

Mammalian medial and lateral hippocampal networks preferentially process spatial- and object-related information, respectively. However, the mechanisms underlying the assembly of such parallel networks during development remain largely unknown. Our study shows that, in mice, complementary expression of cell surface molecules teneurin-3 (Ten3) and latrophilin-2 (Lphn2) in the medial and lateral hippocampal networks, respectively, guides the precise assembly of CA1-to-subiculum connections in both networks. In the medial network, Ten3-expressing (Ten3+) CA1 axons are repelled by target-derived Lphn2, revealing that Lphn2- and Ten3-mediated heterophilic repulsion and Ten3-mediated homophilic attraction cooperate to control precise target selection of CA1 axons. In the lateral network, Lphn2-expressing (Lphn2+) CA1 axons are confined to Lphn2+ targets via repulsion from Ten3+ targets. Our findings demonstrate that assembly of parallel hippocampal networks follows a "Ten3→Ten3, Lphn2→Lphn2" rule instructed by reciprocal repulsions.
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http://dx.doi.org/10.1126/science.abg1774DOI Listing
June 2021

Understanding the human endometrium in the 21st century.

Am J Obstet Gynecol 2021 07 20;225(1):1-2. Epub 2021 May 20.

Igenomix Foundation, INCLIVA Health Research Institute, Valencia, Spain; Department of Obstetrics and Gynecology, University of California, San Francisco, CA; Department of Obstetrics and Gynecology, University of Valencia, Valencia, Spain; Department of Obstetrics and Gynecology, Beth Israel Deaconess Medical Center, Harvard University, Boston, MA. Electronic address:

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http://dx.doi.org/10.1016/j.ajog.2021.04.224DOI Listing
July 2021

Mapping single-cell atlases throughout Metazoa unravels cell type evolution.

Elife 2021 05 4;10. Epub 2021 May 4.

Department of Bioengineering, Stanford University, Stanford, United States.

Comparing single-cell transcriptomic atlases from diverse organisms can elucidate the origins of cellular diversity and assist the annotation of new cell atlases. Yet, comparison between distant relatives is hindered by complex gene histories and diversifications in expression programs. Previously, we introduced the self-assembling manifold (SAM) algorithm to robustly reconstruct manifolds from single-cell data (Tarashansky et al., 2019). Here, we build on SAM to map cell atlas manifolds across species. This new method, SAMap, identifies homologous cell types with shared expression programs across distant species within phyla, even in complex examples where homologous tissues emerge from distinct germ layers. SAMap also finds many genes with more similar expression to their paralogs than their orthologs, suggesting paralog substitution may be more common in evolution than previously appreciated. Lastly, comparing species across animal phyla, spanning sponge to mouse, reveals ancient contractile and stem cell families, which may have arisen early in animal evolution.
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http://dx.doi.org/10.7554/eLife.66747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8139856PMC
May 2021

Corrigendum: Investigating Pregnancy and Its Complications Using Circulating Cell-Free RNA in Women's Blood During Gestation.

Front Pediatr 2021 12;9:680201. Epub 2021 Apr 12.

Departments of Bioengineering and Applied Physics, Stanford University, and Chan Zuckerberg Biohub, Stanford, CA, United States.

[This corrects the article DOI: 10.3389/fped.2020.605219.].
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http://dx.doi.org/10.3389/fped.2021.680201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8065098PMC
April 2021

Understanding how biologic and social determinants affect disparities in preterm birth and outcomes of preterm infants in the NICU.

Semin Perinatol 2021 Jun 24;45(4):151408. Epub 2021 Mar 24.

Department of Pediatrics, Division of Neonatal and Developmental Medicine, Stanford University School of Medicine, 1265 Welch Rd, X157, Stanford, CA 94305-5415, USA.

To understand the disparities in spontaneous preterm birth (sPTB) and/or its outcomes, biologic and social determinants as well as healthcare practice (such as those in neonatal intensive care units) should be considered. Disparities in sPTB have been largely intractable and remain obscure in most cases, despite a myriad of identified risk factors for and causes of sPTB. We still do not know how they lead to the different outcomes at different gestational ages and if they are independent of NICU practices. Here we describe an integrated approach to study the interplay between the genome and exposome, which may drive biochemistry and physiology and lead to health disparities.
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http://dx.doi.org/10.1016/j.semperi.2021.151408DOI Listing
June 2021

The transcriptional landscape of Venezuelan equine encephalitis virus (TC-83) infection.

PLoS Negl Trop Dis 2021 03 31;15(3):e0009306. Epub 2021 Mar 31.

Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, United States of America.

Venezuelan Equine Encephalitis Virus (VEEV) is a major biothreat agent that naturally causes outbreaks in humans and horses particularly in tropical areas of the western hemisphere, for which no antiviral therapy is currently available. The host response to VEEV and the cellular factors this alphavirus hijacks to support its effective replication or evade cellular immune responses are largely uncharacterized. We have previously demonstrated tremendous cell-to-cell heterogeneity in viral RNA (vRNA) and cellular transcript levels during flaviviral infection using a novel virus-inclusive single-cell RNA-Seq approach. Here, we used this unbiased, genome-wide approach to simultaneously profile the host transcriptome and vRNA in thousands of single cells during infection of human astrocytes with the live-attenuated vaccine strain of VEEV (TC-83). Host transcription was profoundly suppressed, yet "superproducer cells" with extremely high vRNA abundance emerged during the first viral life cycle and demonstrated an altered transcriptome relative to both uninfected cells and cells with high vRNA abundance harvested at later time points. Additionally, cells with increased structural-to-nonstructural transcript ratio exhibited upregulation of intracellular membrane trafficking genes at later time points. Loss- and gain-of-function experiments confirmed pro- and antiviral activities in both vaccine and virulent VEEV infections among the products of transcripts that positively or negatively correlated with vRNA abundance, respectively. Lastly, comparison with single cell transcriptomic data from other viruses highlighted common and unique pathways perturbed by infection across evolutionary scales. This study provides a high-resolution characterization of the VEEV (TC-83)-host interplay, identifies candidate targets for antivirals, and establishes a comparative single-cell approach to study the evolution of virus-host interactions.
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http://dx.doi.org/10.1371/journal.pntd.0009306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041203PMC
March 2021

Single-cell transcriptomes of developing and adult olfactory receptor neurons in .

Elife 2021 Feb 8;10. Epub 2021 Feb 8.

Department of Biology, Howard Hughes Medical Institute, Stanford University, Stanford, United States.

Recognition of environmental cues is essential for the survival of all organisms. Transcriptional changes occur to enable the generation and function of the neural circuits underlying sensory perception. To gain insight into these changes, we generated single-cell transcriptomes of olfactory- (ORNs), thermo-, and hygro-sensory neurons at an early developmental and adult stage using single-cell and single-nucleus RNA sequencing. We discovered that ORNs maintain expression of the same olfactory receptors across development. Using receptor expression and computational approaches, we matched transcriptomic clusters corresponding to anatomically and physiologically defined neuron types across multiple developmental stages. We found that cell-type-specific transcriptomes partly reflected axon trajectory choices in development and sensory modality in adults. We uncovered stage-specific genes that could regulate the wiring and sensory responses of distinct ORN types. Collectively, our data reveal transcriptomic features of sensory neuron biology and provide a resource for future studies of their development and physiology.
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http://dx.doi.org/10.7554/eLife.63856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870146PMC
February 2021

Sexual and asexual development: two distinct programs producing the same tunicate.

Cell Rep 2021 01;34(4):108681

Institute for Stem Cell Biology and Regenerative Medicine, and Ludwig Center, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Biology, Stanford University, Hopkins Marine Station, Pacific Grove, CA 93950, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA. Electronic address:

Colonial tunicates are the only chordate that possess two distinct developmental pathways to produce an adult body: either sexually through embryogenesis or asexually through a stem cell-mediated renewal termed blastogenesis. Using the colonial tunicate Botryllus schlosseri, we combine transcriptomics and microscopy to build an atlas of the molecular and morphological signatures at each developmental stage for both pathways. The general molecular profiles of these processes are largely distinct. However, the relative timing of organogenesis and ordering of tissue-specific gene expression are conserved. By comparing the developmental pathways of B. schlosseri with other chordates, we identify hundreds of putative transcription factors with conserved temporal expression. Our findings demonstrate that convergent morphology need not imply convergent molecular mechanisms but that it showcases the importance that tissue-specific stem cells and transcription factors play in producing the same mature body through different pathways.
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http://dx.doi.org/10.1016/j.celrep.2020.108681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949349PMC
January 2021

Noninvasive stratification of nonalcoholic fatty liver disease by whole transcriptome cell-free mRNA characterization.

Am J Physiol Gastrointest Liver Physiol 2021 04 27;320(4):G439-G449. Epub 2021 Jan 27.

Molecular Stethoscope, San Diego, California.

Hepatic fibrosis stage is the most important determinant of outcomes in patients with nonalcoholic fatty liver disease (NAFLD). There is an urgent need for noninvasive tests that can accurately stage fibrosis and determine efficacy of interventions. Here, we describe a novel cell-free (cf)-mRNA sequencing approach that can accurately and reproducibly profile low levels of circulating mRNAs and evaluate the feasibility of developing a cf-mRNA-based NAFLD fibrosis classifier. Using separate discovery and validation cohorts with biopsy-confirmed NAFLD ( = 176 and 59, respectively) and healthy subjects ( = 23), we performed serum cf-mRNA RNA-Seq profiling. Differential expression analysis identified 2,498 dysregulated genes between patients with NAFLD and healthy subjects and 134 fibrosis-associated genes in patients with NAFLD. Comparison between cf-mRNA and liver tissue transcripts revealed significant overlap of fibrosis-associated genes and pathways indicating that the circulating cf-mRNA transcriptome reflects molecular changes in the livers of patients with NAFLD. In particular, metabolic and immune pathways reflective of known underlying steatosis and inflammation were highly dysregulated in the cf-mRNA profile of patients with advanced fibrosis. Finally, we used an elastic net ordinal logistic model to develop a classifier that predicts clinically significant fibrosis (F2-F4). In an independent cohort, the cf-mRNA classifier was able to identify 50% of patients with at least 90% probability of clinically significant fibrosis. We demonstrate a novel and robust cf-mRNA-based RNA-Seq platform for noninvasive identification of diverse hepatic molecular disruptions and for fibrosis staging with promising potential for clinical trials and clinical practice. This work is the first study, to our knowledge, to utilize circulating cell-free mRNA sequencing to develop an NAFLD diagnostic classifier.
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http://dx.doi.org/10.1152/ajpgi.00397.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8238173PMC
April 2021

Single-cell analysis of Schistosoma mansoni identifies a conserved genetic program controlling germline stem cell fate.

Nat Commun 2021 01 20;12(1):485. Epub 2021 Jan 20.

Department of Bioengineering, Stanford University, Stanford, CA, 94305, USA.

Schistosomes are parasitic flatworms causing one of the most prevalent infectious diseases from which millions of people are currently suffering. These parasites have high fecundity and their eggs are both the transmissible agents and the cause of the infection-associated pathology. Given its biomedical significance, the schistosome germline has been a research focus for more than a century. Nonetheless, molecular mechanisms that regulate its development are only now being understood. In particular, it is unknown what balances the fate of germline stem cells (GSCs) in producing daughter stem cells through mitotic divisions versus gametes through meiosis. Here, we perform single-cell RNA sequencing on juvenile schistosomes and capture GSCs during de novo gonadal development. We identify a genetic program that controls the proliferation and differentiation of GSCs. This program centers around onecut, a homeobox transcription factor, and boule, an mRNA binding protein. Their expressions are mutually dependent in the schistosome male germline, and knocking down either of them causes over-proliferation of GSCs and blocks germ cell differentiation. We further show that this germline-specific regulatory program is conserved in the planarian, schistosome's free-living evolutionary cousin, but the function of onecut has changed during evolution to support GSC maintenance.
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http://dx.doi.org/10.1038/s41467-020-20794-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817839PMC
January 2021

Temporal evolution of single-cell transcriptomes of olfactory projection neurons.

Elife 2021 Jan 11;10. Epub 2021 Jan 11.

Department of Biology, Howard Hughes Medical Institute, Stanford University, Stanford, United States.

Neurons undergo substantial morphological and functional changes during development to form precise synaptic connections and acquire specific physiological properties. What are the underlying transcriptomic bases? Here, we obtained the single-cell transcriptomes of olfactory projection neurons (PNs) at four developmental stages. We decoded the identity of 21 transcriptomic clusters corresponding to 20 PN types and developed methods to match transcriptomic clusters representing the same PN type across development. We discovered that PN transcriptomes reflect unique biological processes unfolding at each stage-neurite growth and pruning during metamorphosis at an early pupal stage; peaked transcriptomic diversity during olfactory circuit assembly at mid-pupal stages; and neuronal signaling in adults. At early developmental stages, PN types with adjacent birth order share similar transcriptomes. Together, our work reveals principles of cellular diversity during brain development and provides a resource for future studies of neural development in PNs and other neuronal types.
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http://dx.doi.org/10.7554/eLife.63450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870145PMC
January 2021

Investigating Pregnancy and Its Complications Using Circulating Cell-Free RNA in Women's Blood During Gestation.

Front Pediatr 2020 14;8:605219. Epub 2020 Dec 14.

Departments of Bioengineering and Applied Physics, Stanford University, and Chan Zuckerberg Biohub, Stanford, CA, United States.

In recent years, there have been major advances in the application of non-invasive techniques to predict pregnancy-related complications, for example by measuring cell-free RNA (cfRNA) in maternal blood. In contrast to cell-free DNA (cfDNA), which is already in clinical use to diagnose fetal aneuploidy, circulating RNA levels can correspond with tissue-specific gene expression and provide a snapshot of prenatal health across gestation. Here, we review the physiologic origins of cfRNA and its novel applications and corresponding challenges to monitor fetal and maternal health and predict pregnancy-related complications.
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http://dx.doi.org/10.3389/fped.2020.605219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7767905PMC
December 2020

Differential encoding in prefrontal cortex projection neuron classes across cognitive tasks.

Cell 2021 01 17;184(2):489-506.e26. Epub 2020 Dec 17.

Department of Biology, Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA. Electronic address:

Single-cell transcriptomics has been widely applied to classify neurons in the mammalian brain, while systems neuroscience has historically analyzed the encoding properties of cortical neurons without considering cell types. Here we examine how specific transcriptomic types of mouse prefrontal cortex (PFC) projection neurons relate to axonal projections and encoding properties across multiple cognitive tasks. We found that most types projected to multiple targets, and most targets received projections from multiple types, except PFC→PAG (periaqueductal gray). By comparing Ca activity of the molecularly homogeneous PFC→PAG type against two heterogeneous classes in several two-alternative choice tasks in freely moving mice, we found that all task-related signals assayed were qualitatively present in all examined classes. However, PAG-projecting neurons most potently encoded choice in cued tasks, whereas contralateral PFC-projecting neurons most potently encoded reward context in an uncued task. Thus, task signals are organized redundantly, but with clear quantitative biases across cells of specific molecular-anatomical characteristics.
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http://dx.doi.org/10.1016/j.cell.2020.11.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935083PMC
January 2021

Multiomics Characterization of Preterm Birth in Low- and Middle-Income Countries.

JAMA Netw Open 2020 12 1;3(12):e2029655. Epub 2020 Dec 1.

Department of Pediatrics and Child Health, Aga Khan University, Karachi, Pakistan.

Importance: Worldwide, preterm birth (PTB) is the single largest cause of deaths in the perinatal and neonatal period and is associated with increased morbidity in young children. The cause of PTB is multifactorial, and the development of generalizable biological models may enable early detection and guide therapeutic studies.

Objective: To investigate the ability of transcriptomics and proteomics profiling of plasma and metabolomics analysis of urine to identify early biological measurements associated with PTB.

Design, Setting, And Participants: This diagnostic/prognostic study analyzed plasma and urine samples collected from May 2014 to June 2017 from pregnant women in 5 biorepository cohorts in low- and middle-income countries (LMICs; ie, Matlab, Bangladesh; Lusaka, Zambia; Sylhet, Bangladesh; Karachi, Pakistan; and Pemba, Tanzania). These cohorts were established to study maternal and fetal outcomes and were supported by the Alliance for Maternal and Newborn Health Improvement and the Global Alliance to Prevent Prematurity and Stillbirth biorepositories. Data were analyzed from December 2018 to July 2019.

Exposures: Blood and urine specimens that were collected early during pregnancy (median sampling time of 13.6 weeks of gestation, according to ultrasonography) were processed, stored, and shipped to the laboratories under uniform protocols. Plasma samples were assayed for targeted measurement of proteins and untargeted cell-free ribonucleic acid profiling; urine samples were assayed for metabolites.

Main Outcomes And Measures: The PTB phenotype was defined as the delivery of a live infant before completing 37 weeks of gestation.

Results: Of the 81 pregnant women included in this study, 39 had PTBs (48.1%) and 42 had term pregnancies (51.9%) (mean [SD] age of 24.8 [5.3] years). Univariate analysis demonstrated functional biological differences across the 5 cohorts. A cohort-adjusted machine learning algorithm was applied to each biological data set, and then a higher-level machine learning modeling combined the results into a final integrative model. The integrated model was more accurate, with an area under the receiver operating characteristic curve (AUROC) of 0.83 (95% CI, 0.72-0.91) compared with the models derived for each independent biological modality (transcriptomics AUROC, 0.73 [95% CI, 0.61-0.83]; metabolomics AUROC, 0.59 [95% CI, 0.47-0.72]; and proteomics AUROC, 0.75 [95% CI, 0.64-0.85]). Primary features associated with PTB included an inflammatory module as well as a metabolomic module measured in urine associated with the glutamine and glutamate metabolism and valine, leucine, and isoleucine biosynthesis pathways.

Conclusions And Relevance: This study found that, in LMICs and high PTB settings, major biological adaptations during term pregnancy follow a generalizable model and the predictive accuracy for PTB was augmented by combining various omics data sets, suggesting that PTB is a condition that manifests within multiple biological systems. These data sets, with machine learning partnerships, may be a key step in developing valuable predictive tests and intervention candidates for preventing PTB.
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http://dx.doi.org/10.1001/jamanetworkopen.2020.29655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749442PMC
December 2020

Cerebellar nuclei evolved by repeatedly duplicating a conserved cell-type set.

Science 2020 12;370(6523)

Department of Biology, Stanford University, Stanford, CA 94305, USA.

How have complex brains evolved from simple circuits? Here we investigated brain region evolution at cell-type resolution in the cerebellar nuclei, the output structures of the cerebellum. Using single-nucleus RNA sequencing in mice, chickens, and humans, as well as STARmap spatial transcriptomic analysis and whole-central nervous system projection tracing, we identified a conserved cell-type set containing two region-specific excitatory neuron classes and three region-invariant inhibitory neuron classes. This set constitutes an archetypal cerebellar nucleus that was repeatedly duplicated to form new regions. The excitatory cell class that preferentially funnels information to lateral frontal cortices in mice becomes predominant in the massively expanded human lateral nucleus. Our data suggest a model of brain region evolution by duplication and divergence of entire cell-type sets.
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http://dx.doi.org/10.1126/science.abd5059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8510508PMC
December 2020

Noninvasive characterization of Alzheimer's disease by circulating, cell-free messenger RNA next-generation sequencing.

Sci Adv 2020 Dec 9;6(50). Epub 2020 Dec 9.

Molecular Stethoscope Inc., 3210 Merryfield Row, San Diego, CA 92121, USA.

The lack of accessible noninvasive tools to examine the molecular alterations occurring in the brain limits our understanding of the causes and progression of Alzheimer's disease (AD), as well as the identification of effective therapeutic strategies. Here, we conducted a comprehensive profiling of circulating, cell-free messenger RNA (cf-mRNA) in plasma of 126 patients with AD and 116 healthy controls of similar age. We identified 2591 dysregulated genes in the cf-mRNA of patients with AD, which are enriched in biological processes well known to be associated with AD. Dysregulated genes included brain-specific genes and resembled those identified to be dysregulated in postmortem AD brain tissue. Furthermore, we identified disease-relevant circulating gene transcripts that correlated with the severity of cognitive impairment. These data highlight the potential of high-throughput cf-mRNA sequencing to evaluate AD-related pathophysiological alterations in the brain, leading to precision healthcare solutions that could improve AD patient management.
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http://dx.doi.org/10.1126/sciadv.abb1654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821903PMC
December 2020

A molecular cell atlas of the human lung from single-cell RNA sequencing.

Nature 2020 11 18;587(7835):619-625. Epub 2020 Nov 18.

Department of Biochemistry, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA.

Although single-cell RNA sequencing studies have begun to provide compendia of cell expression profiles, it has been difficult to systematically identify and localize all molecular cell types in individual organs to create a full molecular cell atlas. Here, using droplet- and plate-based single-cell RNA sequencing of approximately 75,000 human cells across all lung tissue compartments and circulating blood, combined with a multi-pronged cell annotation approach, we create an extensive cell atlas of the human lung. We define the gene expression profiles and anatomical locations of 58 cell populations in the human lung, including 41 out of 45 previously known cell types and 14 previously unknown ones. This comprehensive molecular atlas identifies the biochemical functions of lung cells and the transcription factors and markers for making and monitoring them; defines the cell targets of circulating hormones and predicts local signalling interactions and immune cell homing; and identifies cell types that are directly affected by lung disease genes and respiratory viruses. By comparing human and mouse data, we identified 17 molecular cell types that have been gained or lost during lung evolution and others with substantially altered expression profiles, revealing extensive plasticity of cell types and cell-type-specific gene expression during organ evolution including expression switches between cell types. This atlas provides the molecular foundation for investigating how lung cell identities, functions and interactions are achieved in development and tissue engineering and altered in disease and evolution.
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http://dx.doi.org/10.1038/s41586-020-2922-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7704697PMC
November 2020

Senescent cells promote tissue NAD decline during ageing via the activation of CD38 macrophages.

Nat Metab 2020 11 16;2(11):1265-1283. Epub 2020 Nov 16.

Buck Institute for Research on Aging, Novato, CA, USA.

Declining tissue nicotinamide adenine dinucleotide (NAD) levels are linked to ageing and its associated diseases. However, the mechanism for this decline is unclear. Here, we show that pro-inflammatory M1-like macrophages, but not naive or M2 macrophages, accumulate in metabolic tissues, including visceral white adipose tissue and liver, during ageing and acute responses to inflammation. These M1-like macrophages express high levels of the NAD-consuming enzyme CD38 and have enhanced CD38-dependent NADase activity, thereby reducing tissue NAD levels. We also find that senescent cells progressively accumulate in visceral white adipose tissue and liver during ageing and that inflammatory cytokines secreted by senescent cells (the senescence-associated secretory phenotype, SASP) induce macrophages to proliferate and express CD38. These results uncover a new causal link among resident tissue macrophages, cellular senescence and tissue NAD decline during ageing and offer novel therapeutic opportunities to maintain NAD levels during ageing.
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http://dx.doi.org/10.1038/s42255-020-00305-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7908681PMC
November 2020

Persistent transcriptional programmes are associated with remote memory.

Nature 2020 11 11;587(7834):437-442. Epub 2020 Nov 11.

Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA.

The role of gene expression during learning and in short-term memories has been studied extensively, but less is known about remote memories, which can persist for a lifetime. Here we used long-term contextual fear memory as a paradigm to probe the single-cell gene expression landscape that underlies remote memory storage in the medial prefrontal cortex. We found persistent activity-specific transcriptional alterations in diverse populations of neurons that lasted for weeks after fear learning. Out of a vast plasticity-coding space, we identified genes associated with membrane fusion that could have important roles in the maintenance of remote memory. Unexpectedly, astrocytes and microglia also acquired persistent gene expression signatures that were associated with remote memory, suggesting that they actively contribute to memory circuits. The discovery of gene expression programmes associated with remote memory engrams adds an important dimension of activity-dependent cellular states to existing brain taxonomy atlases and sheds light on the elusive mechanisms of remote memory storage.
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http://dx.doi.org/10.1038/s41586-020-2905-5DOI Listing
November 2020

Detection of early stage pancreatic cancer using 5-hydroxymethylcytosine signatures in circulating cell free DNA.

Nat Commun 2020 10 19;11(1):5270. Epub 2020 Oct 19.

Bluestar Genomics, 185 Berry Street, Lobby 4, Suite 210, San Francisco, CA, 94107, USA.

Pancreatic cancer is often detected late, when curative therapies are no longer possible. Here, we present non-invasive detection of pancreatic ductal adenocarcinoma (PDAC) by 5-hydroxymethylcytosine (5hmC) changes in circulating cell free DNA from a PDAC cohort (n = 64) in comparison with a non-cancer cohort (n = 243). Differential hydroxymethylation is found in thousands of genes, most significantly in genes related to pancreas development or function (GATA4, GATA6, PROX1, ONECUT1, MEIS2), and cancer pathogenesis (YAP1, TEAD1, PROX1, IGF1). cfDNA hydroxymethylome in PDAC cohort is differentially enriched for genes that are commonly de-regulated in PDAC tumors upon activation of KRAS and inactivation of TP53. Regularized regression models built using 5hmC densities in genes perform with AUC of 0.92 (discovery dataset, n = 79) and 0.92-0.94 (two independent test sets, n = 228). Furthermore, tissue-derived 5hmC features can be used to classify PDAC cfDNA (AUC = 0.88). These findings suggest that 5hmC changes enable classification of PDAC even during early stage disease.
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http://dx.doi.org/10.1038/s41467-020-18965-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572413PMC
October 2020

A mouse tissue atlas of small noncoding RNA.

Proc Natl Acad Sci U S A 2020 10 25;117(41):25634-25645. Epub 2020 Sep 25.

Department of Bioengineering, Stanford University, Stanford, CA 94305;

Small noncoding RNAs (ncRNAs) play a vital role in a broad range of biological processes both in health and disease. A comprehensive quantitative reference of small ncRNA expression would significantly advance our understanding of ncRNA roles in shaping tissue functions. Here, we systematically profiled the levels of five ncRNA classes (microRNA [miRNA], small nucleolar RNA [snoRNA], small nuclear RNA [snRNA], small Cajal body-specific RNA [scaRNA], and transfer RNA [tRNA] fragments) across 11 mouse tissues by deep sequencing. Using 14 biological replicates spanning both sexes, we identified that ∼30% of small ncRNAs are distributed across the body in a tissue-specific manner with some also being sexually dimorphic. We found that some miRNAs are subject to "arm switching" between healthy tissues and that tRNA fragments are retained within tissues in both a gene- and a tissue-specific manner. Out of 11 profiled tissues, we confirmed that brain contains the largest number of unique small ncRNA transcripts, some of which were previously annotated while others are identified in this study. Furthermore, by combining these findings with single-cell chromatin accessibility (scATAC-seq) data, we were able to connect identified brain-specific ncRNAs with their cell types of origin. These results yield the most comprehensive characterization of specific and ubiquitous small RNAs in individual murine tissues to date, and we expect that these data will be a resource for the further identification of ncRNAs involved in tissue function in health and dysfunction in disease.
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http://dx.doi.org/10.1073/pnas.2002277117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568261PMC
October 2020

Northstar enables automatic classification of known and novel cell types from tumor samples.

Sci Rep 2020 09 17;10(1):15251. Epub 2020 Sep 17.

Department of Bioengineering, Stanford University, Stanford, CA, USA.

Single cell transcriptomics is revolutionising our understanding of tissue and disease heterogeneity, yet cell type identification remains a partially manual task. Published algorithms for automatic cell annotation are limited to known cell types and fail to capture novel populations, especially cancer cells. We developed northstar, a computational approach to classify thousands of cells based on published data within seconds while simultaneously identifying and highlighting new cell states such as malignancies. We tested northstar on data from glioblastoma, melanoma, and seven different healthy tissues and obtained high accuracy and robustness. We collected eleven pancreatic tumors and identified three shared and five private neoplastic cell populations, offering insight into the origins of neuroendocrine and exocrine tumors. Northstar is a useful tool to assign known and novel cell type and states in the age of cell atlases.
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http://dx.doi.org/10.1038/s41598-020-71805-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499423PMC
September 2020

Single-cell transcriptomic atlas of the human endometrium during the menstrual cycle.

Nat Med 2020 10 14;26(10):1644-1653. Epub 2020 Sep 14.

Department of Bioengineering, Stanford University, Stanford, CA, USA.

In a human menstrual cycle the endometrium undergoes remodeling, shedding and regeneration, all of which are driven by substantial gene expression changes in the underlying cellular hierarchy. Despite its importance in human fertility and regenerative biology, our understanding of this unique type of tissue homeostasis remains rudimentary. We characterized the transcriptomic transformation of human endometrium at single-cell resolution across the menstrual cycle, resolving cellular heterogeneity in multiple dimensions. We profiled the behavior of seven endometrial cell types, including a previously uncharacterized ciliated cell type, during four major phases of endometrial transformation, and found characteristic signatures for each cell type and phase. We discovered that the human window of implantation opens with an abrupt and discontinuous transcriptomic activation in the epithelia, accompanied with a widespread decidualization feature in the stromal fibroblasts. Our study provides a high-resolution molecular and cellular characterization of human endometrial transformation across the menstrual cycle, providing insights into this essential physiological process.
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http://dx.doi.org/10.1038/s41591-020-1040-zDOI Listing
October 2020

Cloning antibodies from single cells in pooled sequence libraries by selective PCR.

PLoS One 2020 5;15(8):e0236477. Epub 2020 Aug 5.

Department of Bioengineering, Stanford University, Stanford, California, United States of America.

Antibodies function by binding to antigens. Antibodies must be cloned and expressed to determine their binding characteristics, but current methods for high-throughput antibody sequencing yield antibody DNA pooled from many cells and do not readily permit cloning of antibodies from single B cells. We present a strategy for retrieving and cloning antibody DNA from single cells within a pooled library of cells. Our strategy, called selective PCR for antibody retrieval (SPAR), takes advantage of the unique sequence barcodes attached to individual cDNA molecules during sample preparation to enable specific amplification by PCR of antibody heavy- and light-chain cDNA originating from a single cell. We show through computational analysis that most human antibodies sequenced using typical high-throughput methods can be retrieved using SPAR, and experimentally demonstrate retrieval of full-length antibody variable region cDNA from three cells within pools of ~5,000 cells. SPAR enables rapid low-cost cloning and expression of native human antibodies from pooled single-cell sequence libraries for functional characterization.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0236477PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406036PMC
October 2020
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