Biology subjects

Meyer, K.

Publications and source records attributed to Meyer, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Decoding of YAP levels and dynamics by pluripotency factors

YAP is a transcriptional regulator that controls pluripotency, germ layer specification, and proliferation. Different subsets of YAP target genes are engaged in each physiological setting, but how YAP selectively regulates different effectors in different contexts is not known. Here we use optogenetics to investigate how the levels and dynamics of YAP activation control its pluripotency effectors Oct4 and Nanog. We observe different thresholds for repression of Oct4 and Nanog, enabling differential control of both genes through YAP levels. Pluripotency factors also decode YAP dynamics. Oct4 preferentially responds to oscillatory YAP inputs that mimic endogenous pulsatile YAP dynamics. Using single-cell live imaging of Oct4 transcription and computational-theoretical analysis of transcriptional regulation, we demonstrate that YAP dynamics are decoded by an adaptive change sensor that modulates Oct4 transcription burst frequency. Our results reveal how the levels and timing of YAP activation enable multiplexing of information transmission for key regulators of cellular differentiation and pluripotency.

cell biology↗

bioRxiv2022

Single-molecule identification of the target RNAs of different RNA binding proteins simultaneously in cells

RNA-binding proteins (RBPs) regulate nearly every aspect of mRNA processing and are important regulators of gene expression in cells. However, current methods for transcriptome-wide identification of RBP targets are limited since they examine only a single RBP at a time and since they do not provide information on the individual RNA molecules that are bound by a given RBP. Here, we overcome these limitations by developing TRIBE-STAMP, an approach for single-molecule detection of the target RNAs of two RNA binding proteins simultaneously in cells. We apply TRIBE-STAMP to the cytoplasmic m6A reader proteins YTHDF1, 2, and 3 and discover that individual mRNA molecules can be bound by more than one YTHDF protein throughout their lifetime, providing new insights into the function of YTHDF proteins in cells. TRIBE-STAMP is a highly versatile approach that enables single-molecule analysis of the targets of RBP pairs simultaneously in the same cells.

molecular biology↗

bioRxiv2022

Iron accumulation drives fibrosis, senescence, and the senescence-associated secretory phenotype

Fibrogenesis is part of a normal protective response to tissue injury that can become irreversible and progressive, leading to fatal diseases. Senescent cells are a main driver of fibrotic diseases through their secretome, known as senescence-associated secretory phenotype (SASP). However, the mechanisms involved in the conversion of damaged cells into senescent cells remain incompletely understood. Here, we report that multiple types of fibrotic diseases in mice and humans are characterized by the accumulation of iron. We show that vascular and hemolytic injuries, through the release of iron, are efficient in triggering senescence and fibrosis. Interestingly, the accumulation of iron is an intrinsic property of senescent cells that does not require an abnormal surge in extracellular iron. Upon damage, cells initiate an iron accumulation response with abundant ferritin-bound iron within lysosomes and high levels of labile iron, the latter being a main driver of senescence-associated ROS and SASP. Finally, we demonstrate that detection of iron by magnetic resonance imaging (MRI) is a powerful non-invasive method to assess fibrotic burden in the kidneys of mice and patients with renal fibrosis. Our findings establish a central role for iron accumulation in senescence and fibrogenesis.

physiology↗

bioRxiv2022

Modified E2 glycoprotein of hepatitis C virus enhances pro-inflammatory cytokines and protective immune response in mice

Hepatitis C virus (HCV) is characterized by a high number of chronic cases owing to an impairment of innate and adaptive immune responses. CD81 on the cell surface facilitates HCV entry by interacting with the E2 envelope glycoprotein. On the other hand, CD81/E2 binding on immune related cells may also influence host response outcome to HCV infection. Here, we performed site-specific amino acid substitution in the front layer of E2 sequence to reduce CD81 binding and evaluate HCV candidate vaccine potential. The altered sE2 protein (F442NYT), unlike sE2, displayed a significant reduction in CD81 binding, induced pro-inflammatory cytokines, and repressed anti-inflammatory response in primary monocyte-derived macrophages as antigen presenting cells. Further, sE2F442NYT stimulated CD4+T cell proliferation. Immunization of Balb/c mice with an E1/sE2F442NYT RNA-lipid nanoparticle (LNP) displayed improved IgG1 to IgG2a isotype switching, an increase in HCV pseudotype virus neutralizing antibodies, and resistance to challenge infection with a surrogate recombinant vaccinia virus expressing HCV E1-E2-NS2(aa134-966), unlike parental E1/sE2 immunization. Further investigation on modified E2 antigen for selection as antigen may provide helpful information for HCV vaccine development. One Sentence SummaryReduced HCV E2-CD81 binding and immune response

microbiology↗

bioRxiv2021

Mapping interindividual dynamics of innate immune response at single-cell resolution

Common genetic variants modulate the cellular response to viruses and are implicated in a range of immune pathologies, including infectious and autoimmune diseases. The transcriptional antiviral response is known to vary between infected cells from a single individual, yet how genetic variants across individuals modulate the antiviral response (and its cell-to-cell variability) is not well understood. Here, we triggered the antiviral response in human fibroblasts from 68 healthy donors, and profiled tens of thousands of cells using single-cell RNA-seq. We developed GASPACHO (GAuSsian Processes for Association mapping leveraging Cell HeterOgeneity), the first statistical approach designed to identify dynamic eQTLs across a transcriptional trajectory of cell populations, without aggregating single-cell data into pseudo-bulk. This allows us to uncover the underlying architecture and variability of antiviral response across responding cells, and to identify more than two thousands eQTLs modulating the dynamic changes during this response. Many of these eQTLs colocalise with risk loci identified in GWAS of infectious and autoimmune diseases. As a case study, we focus on a COVID-19 susceptibility locus, colocalised with the antiviral OAS1 splicing QTL. We validated it in blood cells from a patient cohort and in the infected nasal cells of a patient with the risk allele, demonstrating the utility of GASPACHO to fine-map and functionally characterise a genetic locus. In summary, our novel analytical approach provides a new framework for delineation of the genetic variants that shape a wide spectrum of transcriptional responses at single-cell resolution.

genetics↗

bioRxiv2021

Nuclear Factor I in neurons, glia and during the formation of Muller glia-derived progenitor cells in avian, porcine and primate retinas

The regenerative potential of Muller glia (MG) is extraordinary in fish, poor in chick and terrible in mammals. In the chick model, MG readily reprogram into proliferating Muller glia-derived progenitor cells (MGPCs), but neuronal differentiation is very limited. The factors that suppress the neurogenic potential of MGPCs in the chick are slowly being revealed. Isoforms of Nuclear Factor I (NFI) are cell-intrinsic factors that limit neurogenic potential; these factors are required for the formation of MG in the developing mouse retina (Clark et al., 2019) and deletion of these factors reprograms MG into neuron-like cells in mature mouse retina (Hoang et al., 2020). Accordingly, we sought to characterize the patterns of expression NFIs in the developing, mature and damaged chick retina. In addition, we characterized patterns of expression of NFIs in the retinas of large mammals, pigs and monkeys. Using a combination of single cell RNA-sequencing (scRNA-seq) and immunolabeling we probed for patterns of expression. In embryonic chick, levels of NFIs are very low in early E5 (embryonic day 5) retinal progenitor cells (RPCs), up-regulated in E8 RPCs, further up-regulated in differentiating MG at E12 and E15. NFIs are maintained in mature resting MG, microglia and neurons. Levels of NFIs are reduced in activated MG in retinas treated with NMDA and/or insulin+FGF2, and further down-regulated in proliferating MGPCs. However, levels of NFIs in MGPCs were significantly higher than those seen in RPCs. Immunolabeling for NFIA and NFIB closely matched patterns of expression revealed in different types of retinal neurons and glia, consistent with findings from scRNA-seq. In addition, we find expression of NFIA and NFIB through progenitors in the circumferential marginal zone at the far periphery of the retina. We find similar patterns of expression for NFIs in scRNA-seq databases for pig and monkey retinas. Patterns of expression of NFIA and NFIB were validated with immunofluorescence in pig and monkey retinas wherein these factors were predominantly detected in MG and a few types of inner retinal neurons. In summary, NFIA and NFIB are prominently expressed in developing chick retina and by mature neurons and glia in the retinas of chicks, pigs and monkeys. Although levels of NFIs are decreased in chick, in MGPCs these levels remain higher than those seen in neurogenic RPCs. We propose that the neurogenic potential of MGPCs in the chick retina is suppressed by NFIs.

neuroscience↗

bioRxiv2021

Cross-tissue immune cell analysis reveals tissue-specific adaptations and clonal architecture across the human body

Despite their crucial role in health and disease, our knowledge of immune cells within human tissues remains limited. Here, we surveyed the immune compartment of 15 tissues of six deceased adult donors by single-cell RNA sequencing and paired VDJ sequencing. To systematically resolve immune cell heterogeneity across tissues, we developed CellTypist, a machine learning tool for rapid and precise cell type annotation. Using this approach, combined with detailed curation, we determined the tissue distribution of 45 finely phenotyped immune cell types and states, revealing hitherto unappreciated tissue-specific features and clonal architecture of T and B cells. In summary, our multi-tissue approach lays the foundation for identifying highly resolved immune cell types by leveraging a common reference dataset, tissue-integrated expression analysis and antigen receptor sequencing. One Sentence SummaryWe provide an immune cell atlas, including antigen receptor repertoire profiling, across lymphoid and non-lymphoid human tissues.

immunology↗

bioRxiv2021

SARS-CoV-2 spike protein expressing epithelial cells promotes senescence associated secretory phenotype in endothelial cells and increased inflammatory response

Increased mortality in COVID-19 often associates with thrombotic and microvascular complications. We have recently shown that SARS-CoV-2 spike protein promotes inflammatory cytokine IL-6/IL-6R induced trans-signaling responses which modulate MCP-1 expression in human endothelial cells. MCP-1 is secreted as a major component of the senescence associated secretory phenotype (SASP). Virus infected or Spike transfected human pulmonary epithelial cells (A549) exhibited an increase in senescence related marker proteins. TMNK; as a representative human endothelial cell line, when exposed to cell culture supernatant derived from A549 cells expressing SARS-CoV-2 spike protein (Spike CM) exhibited a senescence phenotype with enhanced p16, p21, and SA-{beta}-galactosidase expression. Inhibition of IL-6 trans-signaling by Tocilizumab, prior to exposure of supernatant to endothelial cells, inhibited p16 and p21 induction. Likewise, inhibition of receptor signaling by Zanabrutinib or Brd4 function by AZD5153 also led to limited induction of p16 expression. Senescence lead to an enhanced level of adhesion molecule, ICAM-1 and VCAM-1 in human endothelial cells, and TPH1 attachment by in vitro assay. Inhibition of senescence or SASP function prevented ICAM/VCAM expression and leukocyte attachment. We also observed an increase in oxidative stress in A549 spike transfected and endothelial cells exposed to Spike CM. ROS generation in TMNK was reduced after treatment with the IL-6 specific inhibitor Tociliximab, and with the specific inhibitors Zanabrutinib and AZD5153. Taken together, we identified that the exposure of human endothelial cells to cell culture supernatant derived from SARS-CoV-2 spike protein expression displayed cellular senescence markers leading to enhanced leukocyte adhesion with coronary blockade potential.

immunology↗

bioRxiv2021

CRISPR-Targeted CAR Gene Insertion Using Cas9/RNP and AAV6 Enhances Anti-AML Activity of Primary NK Cells

Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. Recently, we described an efficient method for gene targeting in NK cells using Cas9/ribonucleoprotein (RNP) complexes. Here we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus using a wide variety of homology arms for homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. For proof-of-concept, we generated mCherry-expressing primary NK cells and determined that sc vectors with 300bp homology arms had optimal transduction efficiency. Then, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3{zeta} and NKG2D/2B4+CD3{zeta}) and expanded them on CSTX002 feeder cells. Expansion kinetics were unaltered and the expanded NK cells maintained high CAR expression (mean 68% CAR+). The CD33-CAR-NK cells showed increased activation markers and enhanced antileukemic activity with improved killing kinetics against CD33-positive acute myeloid leukemia (AML) cell lines and primary samples. Using targeted sequencing we demonstrated the accuracy of CAR gene insertion in human primary NK cells genome. Site-directed insertion using RNP and scAAV6 is an efficient method for stable genetic transfer into primary NK cells that has broad potential for fundamental discovery and therapeutic applications.

immunology↗

bioRxiv2020

Poised cell circuits in human skin are activated in disease

The human skin confers biophysical and immunological protection through a complex cellular network that is established early in development. We profiled ~500,000 single cells using RNA-sequencing from healthy adult and developing skin, and skin from patients with atopic dermatitis and psoriasis. Our findings reveal a predominance of innate lymphoid cells and macrophages in developing skin in contrast to T cells and migratory dendritic cells in adult skin. We demonstrate dual keratinocyte differentiation trajectories and activated cellular circuits comprising vascular endothelial cells mediating immune cell trafficking, disease-specific clonally expanded IL13/IL22 and IL17A/F-expressing lymphocytes, epidermal IL23-expressing dendritic cells and inflammatory keratinocytes in disease. Our findings provide key insights into the dynamic cellular landscape of human skin in health and disease. One Sentence SummarySingle cell atlas of human skin reveals cell circuits which are quantitatively and qualitatively reconfigured in inflammatory skin disease.

immunology↗

bioRxiv2020

AAV9 gene therapy restores lifespan and treats pathological and behavioral abnormalities in a mouse model of CLN8-Batten disease

CLN8 disease is a rare form of neuronal ceroid lipofuscinosis caused by biallelic mutations in the CLN8 gene, which encodes a transmembrane endoplasmic reticulum protein involved in trafficking of lysosomal enzymes. CLN8 disease patients present with myoclonus, tonic-clonic seizures, and progressive declines in cognitive and motor function, with many cases resulting in premature death early in life. There are currently no treatments that can cure the disease or substantially slow disease progression. Using a mouse model of CLN8 disease, we tested the safety and efficacy of an intracerebroventricularly (ICV)-delivered self-complementary AAV9 (scAAV9) gene therapy vector driving expression of human CLN8. A single neonatal injection was safe and well-tolerated, resulting in robust transgene expression throughout the brain and spinal cord from 4 to 24 months, reducing histopathological and behavioral hallmarks of the disease and completely restoring lifespan from 10 months in untreated animals to beyond 24 months of age in treated animals. These results demonstrate, by far, the most successful rescue reported in an animal model of CLN8 disease, and supports gene therapy as a promising therapeutic strategy for this disorder.

neuroscience↗

bioRxiv2019

Highly efficient site-directed gene insertion in primary human natural killer cells using homologous recombination and CRISPaint delivered by AAV

Human peripheral blood natural killer (NK) cells have strong antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. However, genetic modification of NK cells has been challenging due to the high expression of innate sensing mechanisms for viral nucleic acids. Recently, we described an efficient vector-free method using Cas9/ribonucleoprotein complexes for gene deletion in NK cells. Here, we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a user-defined locus using homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. Using these approaches, we identified scAAV6 as the superior serotype for successful generation of stable mCherry-expressing primary NK cells (up to 89%). To maximize transgene packaging in HR-directed gene insertion, we identified minimum optimal homology arm lengths of 300bp for the flanking region of the Cas9-targeting site. Lastly, we demonstrate that mCherry positive NK cells can be expanded to large numbers using feeder cells expressing membrane-bound IL-21. This efficient method for site-directed insertion of genetic material into NK cells has broad potential for basic discovery and therapeutic applications for primary NK cells.

immunology↗

bioRxiv2019

Decoding the development of the blood and immune systems during human fetal liver haematopoiesis

Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells/multipotent progenitors (HSC/MPPs), yet remains poorly defined in humans. Using single cell transcriptome profiling of ~133,000 fetal liver and ~65,000 fetal skin and kidney cells, we identify the repertoire of blood and immune cells in first and early second trimesters of development. From this data, we infer differentiation trajectories from HSC/MPPs, and evaluate the impact of tissue microenvironment on blood and immune cell development. We predict coupling of mast cell differentiation with erythro-megakaryopoiesis and identify physiological erythropoiesis in fetal skin. We demonstrate a shift in fetal liver haematopoietic composition during gestation away from being erythroid-predominant, accompanied by a parallel change in HSC/MPP differentiation potential, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a valuable reference for understanding and harnessing the therapeutic potential of HSC/MPPs.

developmental biology↗

bioRxiv2019

Association analysis of loci implied in “buffering” epistasis

The existence of buffering mechanisms is an emerging property of biological networks, and this results in the possible existence of \"buffering\" loci, that would allow buildup of robustness through evolution. So far, there are no explicit methods to find loci implied in buffering mechanisms. However, buffering can be seen as interaction with genetic background. Here we develop this idea into a tractable model for quantitative genetics, in which the buffering effect of one locus with many other loci is condensed into a single (statistical) effect, multiplicative on the total (statistical) additive genetic effect. This allows easier interpretation of the results, and it also simplifies the problem of detecting epistasis from quadratic to linear in the number of loci. Armed with this formulation, we construct a linear model for genome-wide association studies that estimates, and declares significance, of multiplicative epistatic effects at single loci. The model has the form of a variance components, norm reaction model and likelihood ratio tests are used for significance. This model is a generalization and explanation of previous ones. We then test our model using bovine data: Brahman and Tropical Composite animals, phenotyped for body weight at yearling and genotyped up to [~]770,000 Single Nucleotide Polymorphisms (SNP). After association analysis and based on False Discovery Rate rules, we find a number of loci with buffering action in one, the other, or both breeds; these loci do not have significant statistical additive effect. Most of these loci have been reported in previous studies, either with an additive effect, or as footprints of selection. We identify epistatic SNPs present in or near genes encoding for proteins that are functionally enriched for peptide activity and transcription factors reported in the context of signatures of selection in multi-breed cattle population studies. These include loci known to be associated with coat color, fertility and adaptation to tropical environments. In these populations we found loci that have a non-significant statistical additive effect but a significant epistatic effect. We argue that the discovery and study of loci associated with buffering effects allows attacking the difficult problems, among others, of release of maintenance variance in artificial and natural selection, of quick adaptation to the environment, and of opposite signs of marker effects in different backgrounds. We conclude that our method and our results generate promising new perspectives for research in evolutionary and quantitative genetics based on the study of loci that buffer effect of other loci.

genetics↗

bioRxiv2019

Metabolic control of YAP via the acto-myosin system during liver regeneration

The mechanisms of organ size control remain poorly understood. A key question is how cells collectively sense the overall status of a tissue. We addressed this problem focusing on mouse liver regeneration, which is controlled by Hippo signalling. Using digital tissue reconstruction and quantitative image analysis, we found that the apical surface of hepatocytes forming the bile canalicular network expands concomitant with an increase of F-actin and phospho-Myosin, to compensate an overload of bile acids. Interestingly, these changes are sensed by the Hippo transcriptional co-activator YAP, which localizes to the apical F-actin-rich region and translocates to the nucleus in dependence of the acto-myosin system. This mechanism tolerates moderate bile acid fluctuations under tissue homeostasis, but activates YAP in response to sustained bile acid overload. Using an integrated biophysical-biochemical model of bile pressure and Hippo signalling, we explained this behaviour by the existence of a mechano-sensory mechanism that activates YAP in a switch-like manner. We propose that the apical surface of hepatocytes acts as a self-regulatory mechano-sensory system that responds to critical levels of bile acids as readout of tissue status.

systems biology↗

bioRxiv2018

Liquid-crystal organization of liver tissue

Functional tissue architecture originates by self-assembly of distinct cell types, following tissue-specific rules of cell-cell interactions. In the liver, a structural model of the lobule was pioneered by Elias in 1949. This model, however, is in contrast with the apparent random 3D arrangement of hepatocytes. Since then, no significant progress has been made to derive the organizing principles of liver tissue. To solve this outstanding problem, we computationally reconstructed 3D tissue geometry from microscopy images and analyzed it applying soft-condensed-matter-physics concepts. Surprisingly, analysis of the spatial organization of cell polarity revealed that hepatocytes are not randomly oriented but follow a long-range liquid-crystal order. This does not depend exclusively on hepatocytes receiving instructive signals by endothelial cells as generally assumed, since silencing Integrin-{beta}1 disrupted both liquid-crystal order and organization of the sinusoidal network. Our results suggest that bi-directional communication between hepatocytes and sinusoids underlies the self-organization of liver tissue.

biophysics↗

bioRxiv2018

A probabilistic model to identify the core microbial community

The core microbial community has been hypothesized to have essential functions ranging from maintaining health in animals to protection against plant disease. However, the identification of the core microbial community is frequently based on arbitrary thresholds, selecting only the most abundant microorganisms. Here, we developed and tested an approach to identify the core community based on a probabilistic model. The Poisson distribution was used to identify OTUs with a probable occurrence in every sample of a given dataset. We identified the core communities of four extensive microbial datasets, and compared the results with conventional, but arbitrary, methods. The datasets were composed of the microbiomes of humans (tongue, gut, and skin), mice (gut), plant (grapevine) tissue, and the maize rhizosphere. Our proposed method revealed core microbial communities with higher richness and diversity than those previously described. This method also includes a greater number of rare taxa in the core, which are often neglected by arbitrary threshold methods. We demonstrated that our proposed method revels a probable core microbial community for each different habitat, which extend our knowledge about shared microbial communities. Our proposed method may help the next steps proving the essential functions of core microbial communities.\n\nOriginality-Significance StatementMore rigorous and less arbitrary statistical methods could increase knowledge regarding the role of microorganisms and their interactions. Here, we suggest a probabilistic method to identify the microbial core community across systems. Our method identifies a large proportion of the rare community that likely belongs to the microbial core community, which was not identified by conventional methods. Our probabilistic model is a non-arbitrary approach to defining the microbial core community, which may help in the next step of the microbial core community studies.

microbiology↗

bioRxiv2018

Fast Batch Alignment of Single Cell Transcriptomes Unifies Multiple Mouse Cell Atlases into an Integrated Landscape

Increasing numbers of large scale single cell RNA-Seq projects are leading to a data explosion, which can only be fully exploited through data integration. Therefore, efficient computational tools for combining diverse datasets are crucial for biology in the single cell genomics era. A number of methods have been developed to assist data integration by removing technical batch effects, but most are computationally intensive. To overcome the challenge of enormous datasets, we have developed BBKNN, an extremely fast graph-based data integration method. We illustrate the power of BBKNN for dimensionalityreduced visualisation and clustering in multiple biological scenarios, including a massive integrative study over several murine atlases. BBKNN successfully connects cell populations across experimentally heterogeneous mouse scRNA-Seq datasets, which reveals global markers of cell type and organspecificity and provides the foundation for inferring the underlying transcription factor network. BBKNN is available at https://github.com/Teichlab/bbknn.

bioinformatics↗