Biology subjects

Yang, A.

Publications and source records attributed to Yang, A..

At least 91 records · Page 5Linked to original sources

bioRxiv2022

Inter-gastruloid heterogeneity revealed by single cell transcriptomics time course: implications for organoid based perturbation studies

Recent advances in organoid and genome editing technologies are allowing for perturbation experiments at an unprecedented scale. However, before doing such experiments it is important to understand the gene expression profile in each of the organoids cells, as well as how much heterogeneity there is between individual organoids. Here we characterise an organoid model of mouse gastrulation called gastruloids using single cell RNA-sequencing of individual organoids at half-day intervals between day 3 and day 5 of differentiation (roughly corresponding to E6.5-E8.75 in vivo). Our study reveals multiple differentiation trajectories that have hitherto not been characterised in gastruloids. Intriguingly, we observe that individual gastruloids displayed a strong bias towards producing either mesodermal (largely somitic) or ectodermal (specifically neural) cell types. This bifurcation is already seen at the earliest sampled time point, and is characterised by increased activity of WNT-associated pathways in mesodermally-biased gastruloids as compared to neurally-biased gastruloids. Notably, at day 5, mesodermal gastruloids show an increase in the proportion of neural cells, while neural gastruloids do not produce notably more mesodermal cells. This is in line with previous studies on how the balance between these cell types is regulated. We demonstrate using in silico simulations that without proper understanding of the inter-organoid heterogeneity, perturbation experiments have either very high false positive or negative rates, depending on the statistical model used. Thus in future studies, modelling of inter-organoid heterogeneity will be crucial when designing organoid-based perturbation studies. HighlightsO_LIA single cell RNA-sequencing time course of day 3 to day 5 mouse gastruloids reveals multiple mesodermal and neural differentiation trajectories hitherto uncharacterised in gastruloids C_LIO_LISingle gastruloid, single cell RNA-sequencing of mouse gastruloids reveals that gastruloids are either mesodermally- or neurally-biased C_LIO_LIThe two classes of gastruloid arise from differences in response strength to the WNT-agonist chiron C_LIO_LIAt day 5, mesodermal gastruloids start making more neural cells, while neural gastruloids do not make more mesodermal cells, aligning with previously studied in vivo feedback loops C_LIO_LIWe show using simulations that understanding interorganoid heterogeneity is a crucial consideration in the design and analysis of well-powered organoid-based perturbation studies C_LI

genomics↗

bioRxiv2022

Development of allosteric, selective cyclin-dependent kinase 2 (CDK2) inhibitors that are negatively cooperative with cyclin binding and show potential as contraceptive agents

Compared to most ATP-site kinase inhibitors, small molecules that target an allosteric pocket have the potential for improved selectivity due to the often observed lower structural similarity at these distal sites. Despite their promise, relatively few examples of structurally confirmed, high-affinity allosteric kinase inhibitors exist. Cyclin-dependent kinase 2 (CDK2) is a target for many therapeutic indications, including non-hormonal contraception.1 However, an inhibitor against this kinase with exquisite selectivity has not reached the market because of the structural similarity between CDKs.1-2 In this paper, we describe the development and mechanism of action of new type III inhibitors that bind CDK2 with nanomolar affinity, making them the highest affinity, structurally confirmed allosteric CDK inhibitors reported. Notably, these anthranilic acid inhibitors exhibit a strong negative cooperative relationship with cyclin binding, which remains an underexplored mechanism for CDK2 inhibition. Furthermore, the binding profile of these compounds in both biophysical and cellular assays demonstrate the promise of this series for further development into a therapeutic selective for CDK2 over highly similar kinases like CDK1. The potential of these inhibitors as efficacious contraceptive agents is seen by incubation with mouse testicular explants, where they recapitulate Cdk2-/- and Spdya-/- phenotypes.

pharmacology and toxicology↗

bioRxiv2022

Development and Application of a Novel Simple Sequence Repeat Mining Algorithm Based on Regular Expression

Simple sequence repeats (SSRs) are molecular genetic markers that are powerful tools in genomics studies; SSR markers are routinely mined as a part of genetic workflows. Here, we developed a novel SSR mining algorithm based on regular expression that can reduce the complexity of commonly used SSR mining software. We used the following SSR mining regular expression: ({i, j}?) (\1) {k}, where i and j denote the minimum and maximum lengths of the motifs of the SSR sequence, respectively, and k is the minimum number of repeat motifs. From this SSR mining algorithm, we developed an SSR sequence analysis software (named "regexSSRw") that is capable of mining eligible SSR loci from FASTA format sequences; regexSSRw can be accessed at https://github.com/renm79/rgxSSRw. This SSR mining algorithm can aid a range of applications, from being used by programmers in the development of SSR mining software to being implemented by scholars into their SSR marker workflow.

bioinformatics↗

bioRxiv2022

A Cambrian soft-bodied conical animal illustrates the origins of lophophorate phyla

The origin and early evolution of lophotrochozoans remain a difficult but crucial issue in reconstructing metazoan phylogeny. Exceptionally preserved fossils have provided hopeful information for resolving this lophotrochozoan problem. Here we identify that Conicula striata, a soft-bodied conical animal from the early Cambrian Chengjiang Lagerstatte, has mosaic characteristics of different lophophorate phyla. C. striata possesses a phoronid-like vermiform trunk housing a U-shaped gut and a coiled lophophore comprising [≥]12 arms with numerous tentacles, but it also bears brachiopod-like features including an undivided mantle enclosing lophophore and a pedicle-like protrusion on annulated trunk. Phylogenetic analysis retrieves C. striata as a total-group lophophorate and as an intermediate taxon between Phoronida and Brachiopoda. This suggests that the bivalved architecture of brachiopods originated from an undivided mantle in a phoronid-like ancestor, and that the ancestral vermiform trunk became reduced during the origin of Brachiopoda, illuminating the origins of body plans in the lophophorate phyla.

paleontology↗

bioRxiv2022

TENT2, TUT4, and TUT7 selectively regulate miRNA sequence and abundance

TENTs generate miRNA isoforms by 3 tailing. However, little is known about how tailing regulates miRNA function. Here, we generated isogenic HEK293T cell lines in which TENT2, TUT4 and TUT7 were knocked out individually or in combination. Together with rescue experiments, we characterized TENT-specific effects by deep sequencing, Northern blot and in vitro assays. We found that 3 tailing is not random but highly specific. In addition to its known adenylation, TENT2 contributes to guanylation and uridylation on mature miRNAs. TUT4 uridylates most miRNAs whereas TUT7 is dispensable. Removing adenylation has a marginal impact on miRNA levels. By contrast, abolishing uridylation leads to dysregulation of a set of miRNAs. Besides let-7, miR-181b and miR-222 are negatively regulated by TUT4/7 via distinct mechanisms while the miR-888 cluster is upregulated specifically by TUT7. Our results uncover the selective actions of TENTs in generating 3 isomiRs and pave the way to investigate their functions.

molecular biology↗

bioRxiv2022

CRISPR-LRS for mapping transgenes in the mouse genome

Microinjected transgenes, including bacterial artificial chromosomes (BACs), insert randomly in the mouse genome. Traditional methods of mapping a transgene are challenging, thus complicating breeding strategies and the accurate interpretation of phenotypes, particularly when a transgene disrupts critical coding or noncoding sequences. Here, we introduce CRISPR-Cas9 long-read sequencing (CRISPR-LRS) to ascertain transgene integration locus and estimated copy number. This method revealed integration loci for both a BAC and Cre-driver line, and estimated the copy numbers for two other BAC mouse lines. CRISPR-LRS offers an easy approach to establish robust breeding practices and accurate phenotyping of most any transgenic mouse line.

genetics↗

bioRxiv2021

A reference induced pluripotent stem cell line for large-scale collaborative studies

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate iPSC lines and deeply characterised their genetic properties using whole genome sequencing, their genomic stability upon CRISPR/Cas9-based gene editing, and their phenotypic properties including differentiation to commonly-used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and hundreds of its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field. SummaryThe authors of this collaborative study deeply characterized human induced pluripotent stem cell (iPSC) lines to rationally select a clonally-derived cell line that performs well across multiple modalities. KOLF2.1J was identified as a candidate reference cell line based on single-cell analysis of its gene expression in the pluripotent state, whole genome sequencing, genomic stability after highly efficient CRISPR-mediated gene editing, integrity of the p53 pathway, and the efficiency with which it differentiated into multiple target cell populations. Since it is deeply characterized and can be readily acquired, KOLF2.1J is an attractive reference cell line for groups working with iPSCs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/472643v6_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@cef106org.highwire.dtl.DTLVardef@31d5d8org.highwire.dtl.DTLVardef@1ce6d8corg.highwire.dtl.DTLVardef@17a19d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

Fruit flies subjected to TBI exhibit genotype-dependent changes in seizure incidence and duration

Traumatic brain injury (TBI) is common and often debilitating. One complication following TBI is post-traumatic seizures (PTS). However, there is a poor understanding of PTS, in part, because it is challenging to model. We sought to develop a fly model of PTS. We used a high-impact trauma (HIT) device to inflict TBI and followed this with behavioral, bang-testing methods to assess seizure behavior. Our results showed PTS incidence was sensitive to genetic background. We also found seizure duration was most pronounced at 30 minutes after injury. Our findings support the efficacy of our fly model for coarse screening of seizure behavior. We expect this model will be useful in first-pass screens intended to identify modifiers of seizure risk following TBI.

neuroscience↗

bioRxiv2021

Exploration of 1 BAY 11-7082 as a novel antibiotic

Exposure of the Gram-negative pathogen Pseudomonas aeruginosa to sub-inhibitory concentrations of antibiotics increases formation of biofilms. We exploited this phenotype to identify molecules with potential antimicrobial activity in a biofilm-based high-throughput screen. The anti-inflammatory compound BAY 11-7082 induced dose-dependent biofilm stimulation, indicative of antibacterial activity. We confirmed that BAY 11-7082 inhibits growth of P. aeruginosa and other priority pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). We synthesized 27 structural analogues, including a series based on the related scaffold 3-(phenylsulfonyl)-2-pyrazinecarbonitrile (PSPC), 10 of which displayed increased anti-Staphylococcal activity. Because the parent molecule inhibits the NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome, we measured the ability of select analogues to reduce IL-1{beta} production in mammalian macrophages, identifying minor differences in the structure-activity relationship for the anti-inflammatory and antibacterial properties of this scaffold. Although we could evolve stably resistant MRSA mutants with cross resistance to BAY 11-7082 and PSPC, their lack of shared mutations suggested that the two molecules could have multiple targets. Finally, we showed that BAY 11-7082 and its analogues potentiate the activity of penicillin G against MRSA, suggesting that this scaffold may serve as an interesting starting point for the development of antibiotic adjuvants. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/462244v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1387110org.highwire.dtl.DTLVardef@86f1ddorg.highwire.dtl.DTLVardef@170edaborg.highwire.dtl.DTLVardef@103a36d_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2021

Germinal center activity and B cell maturation promote protective antibody responses against Plasmodium pre-erythrocytic infection

Blocking Plasmodium, the causative agent of malaria, at the asymptomatic pre-erythrocytic stage would abrogate disease pathology and prevent transmission. Rodent-infectious species of Plasmodium such as P. yoelii (Py) serve as key tools to study vaccine efficacy and disease biology in immune-competent experimental animals. Here we evaluated the differences in vaccine-elicited humoral immunity in two widely used, and vastly diverged, inbred mouse strains, BALB/cJ and C57BL/6J, and identified immunological factors associated with protection. We vaccinated with Py circumsporozoite protein (CSP), the major surface antigen on the sporozoite, and evaluated protective efficacy after mosquito bite challenge. Vaccination achieved 60% sterile protection and otherwise delayed blood stage patency in BALB/cJ mice, whereas; all C57BL/6J mice were infected similar to controls. Interestingly, protection was mediated by antibodies, and could be passively transferred from immunized BALB/cJ mice into naive C57BL/6J. Dissection of the underlying immunological features of protection revealed early deficits in antibody titers and polyclonal avidity in C57BL/6J mice. Additionally, PyCSP-vaccination in BALB/cJ induced a significantly higher proportion of antigen-specific B-cells and class-switched memory B-cell (MBCs) populations than in C57BL/6J mice. Strikingly, C57BL/6J mice also had markedly fewer germinal center experienced, CSP-specific class-switched MBCs compared to BALB/cJ mice. Analysis of the IgG {gamma} chain repertoires by next generation sequencing in PyCSP-specific memory B-cell repertoires also revealed higher somatic hypermutation rates in BALB/cJ mice than in C57BL/6J mice. These findings indicate that BALB/cJ mice achieved higher levels of B cell maturation in response to vaccination with PyCSP, which likely enabled the development of protective antibody responses. Overall, our study indicates that germinal center activity and B cell maturation are key processes in the development of vaccine-elicited protective antibodies against CSP.

immunology↗

bioRxiv2021

Robust de novo design of protein binding proteins from target structural information alone

The design of proteins that bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains an outstanding challenge. We describe a general solution to this problem which starts with a broad exploration of the very large space of possible binding modes and interactions, and then intensifies the search in the most promising regions. We demonstrate its very broad applicability by de novo design of binding proteins to 12 diverse protein targets with very different shapes and surface properties. Biophysical characterization shows that the binders, which are all smaller than 65 amino acids, are hyperstable and bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of four of the binder-target complexes, and all four are very close to the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein-protein interactions, and should guide improvement of both. Our approach now enables targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.

synthetic biology↗

bioRxiv2021

Flexible pri-miRNA structures enable tunable production of 5' isomiRs

Drosha cleavage of a pri-miRNA defines mature microRNA sequence. Drosha cleavage at alternative positions generates 5 isoforms (isomiRs) which have distinctive functions. To understand how pri-miRNA structures influence Drosha cleavage, we performed a systematic analysis of the maturation of endogenous pri-miRNAs and their variants both in vitro and in vivo. We show that, in addition to previously known features, the overall structural flexibility of pri-miRNA impacts Drosha cleavage fidelity. Internal loops and nearby G{middle dot}U wobble pairs on the pri-miRNA stem induce the use of non-canonical cleavage sites by Drosha, resulting in 5 isomiR production. By analyzing patient data deposited in The Cancer Genome Atlas, we provide evidence that alternative Drosha cleavage of pri-miRNAs is a tunable process that responds to the level of pri-miRNA-associated RNA-binding proteins. Together, our findings reveal that Drosha cleavage fidelity can be modulated by altering pri-miRNA structure, a potential mechanism underlying 5 isomiR biogenesis in tumors. HIGHLIGHTSO_LIFlexible pri-miRNA structures lead to 5 isomiR production C_LIO_LIInternal loops and G{middle dot}U pairs of pri-miRNA contribute to alternative Drosha cleavages C_LIO_LIAlternative Drosha cleavage results in 5 isomiRs from both strands of pre-miRNAs C_LIO_LI5 isomiR production is upregulated by pri-miRNA-associated RBPs in cancers C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/456839v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@cab9aorg.highwire.dtl.DTLVardef@1d613b1org.highwire.dtl.DTLVardef@1a6e200org.highwire.dtl.DTLVardef@13f7607_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

A connectome-based neuromarker of the non-verbal number acuity and arithmetic skills

The approximate number system (ANS) is vital for survival and reproduction in animals and crucial in constructing abstract mathematical abilities in humans. Most previous neuroimaging studies focused on identifying discrete brain regions responsible for the ANS and characterizing their functions in numerosity perception. However, there lacks a neuromarker to characterize an individuals ANS acuity, especially one based on the whole-brain functional connectivity (FC). Here, we identified a distributed brain network (i.e., numerosity network) using a connectome-based predictive modeling (CPM) analysis on the resting-state functional magnetic resonance imaging (rs-fMRI) data based on a large sample size. The summed strength of all FCs within the numerosity network could reliably predict individual differences of the ANS acuity in behavior. Furthermore, in an independent dataset from the Human Connectome Project (HCP), we found that the summed FC strength within the numerosity network could also predict individual differences in arithmetic skills. Our findings illustrate that the numerosity network we identified could be an applicable neuromarker of the non-verbal number acuity and might serve as the neural basis underlying the known link between the non-verbal number acuity and mathematical abilities.

neuroscience↗

bioRxiv2021

Surveillance of in situ tumor arrays reveals early environmental control of cancer immunity

The immune phenotype of a tumor is a key predictor of its response to immunotherapy1-4. Patients who respond to immune checkpoint blockade generally present with tumors that are infiltrated by activated T cells, a tumor-immune phenotype referred to as immune inflamed5-7. However, not all immune inflamed tumors respond to therapy, and in addition the majority of patients presents with tumors that lack T cells ( immune desert) or that exclude T cells in the periphery of the tumor islet ( immune excluded)8. Despite the importance of these tumor-immune phenotypes in patients, little is known about their development, heterogeneity or dynamics due to an inability to model these features pre-clinically. Here, we describe an approach designated STAMP (skin tumor array by micro-poration), which combines in vivo noninvasive, high-throughput time-lapse imaging with excisional biopsies and next generation sequencing to characterize the establishment of the immunological niche and follow its evolution during immunotherapy. STAMP involves the seeding of dozens to hundreds clonal tumors in the superficial dermis of a single mouse ear that can be visualized in situ over weeks to months. Using this approach, we found that genetically identical tumors could display surprisingly different immune phenotypes. Although individual tumors of the same array were populated by the same T cell clonotypes, regression or progression of individual tumors were associated with distinct patterns of spatial organization of the T cells. In situ imaging of 14K tumors revealed that immune phenotypes were not static over-time but could rather evolve with tumor growth and response to treatment. Therapy-induced or spontaneous early conversion to the immune inflamed phenotype correlated with tumor regression and enhanced cytotoxic T cell activity. Therefore, STAMP provides a flexible approach to study the relationship between tumor evolution, immune cell dynamics, and tumor microenvironment with therapeutic response.

cancer biology↗

bioRxiv2021

The Mediator tail module cooperates with proneural factors to determine fate identity in Glioblastoma.

Glioblastoma stem cells (GSCs) exhibit latent neuronal lineage differentiation potential governed by the proneural transcription factor Achaete-scute homolog 1 (ASCL1) and harnessing and promoting terminal neuronal differentiation has been proposed as a novel therapeutic strategy. Here, using a genome-wide CRISPR suppressor screen we identified genes required for ASCL1-mediated neuronal differentiation. This approach revealed a specialized function of the Mediator complex tail module and of its subunits MED24 and MED25 for this process in GSCs, human fetal neural stem cells and pluripotent stem cells. We show that upon induction of neuronal differentiation MED25 is recruited to genomic loci co-occupied by ASCL1 to regulate neurogenic gene expression programs. MED24 and MED25 are sufficient to induce neuronal differentiation in GSC cultures and to mediate neuronal differentiation in multiple contexts. Collectively our data expand our understanding of the mechanisms underlying directed terminal neuronal differentiation in brain tumor stem cells and point to a unique function of the Mediator tail in neuronal reprogramming.

cell biology↗

bioRxiv2021

METTL9 regulates N1-histidine methylation of zinc transporters to promote tumor growth

Methyltransferase like 9 (Mettl9) is a member of the methyltransferase like protein family which is characterized by the presence of binding domains for S-adenosyl methionine, (SAM), a co-substrate for methylation reactions. Despite METTL9 is predicted to be a methyltransferase, its enzymatic activity, substrate specificities and biological functions are still poorly characterized. In this study, we revealed a tumor-promoting role for METTL9. We found that deletion of Mettl9 in tumor cells suppresses tumor growth and elicits potent anti-tumor immunity. Mechanistically, METTL9 is a N1-histidine methyltransferase which methylates the histidine residues of a x-His-x-His (xHxH) motif on the substrates. This motif is found extensively in zinc transporter families SLC39s and SLC30s, particularly in SLC39A7. Deletion of Mettl9 impairs cytoplasmic zinc homeostasis, resulting in an altered gene expression program with increased endoplasmic reticulum (ER) stress and reduced cell cycle. Mutation of key METTL9 catalyzed methylhistidine residues of SLC39A7 impairs cytoplasmic zinc homeostasis and affects cell growth as well. Notably, METTL9 expression is increased in some cancer types and its higher expression is associated with worse clinical outcomes, particularly in liver and pancreatic cancer. In summary, our work identified METTL9 as a potential new oncogene and its mediated methylation is of regulatory importance. Identifying selective and potent small-molecule inhibitors of METTL9 could thus represent novel therapeutic strategy for anti-proliferative cancer drugs.

cancer biology↗

bioRxiv2020

Genetic determinants of COVID-19 drug efficacy revealed by genome-wide CRISPR screens

Immunomodulatory agents dexamethasone and colchicine, antiviral drugs remdesivir, favipiravir and ribavirin, as well as antimalarial drugs chloroquine phosphate and hydroxychloroquine are currently used in the combat against COVID-191-16. However, whether some of these drugs have clinical efficacy for COVID-19 is under debate. Moreover, these drugs are applied in COVID-19 patients with little knowledge of genetic biomarkers, which will hurt patient outcome. To answer these questions, we designed a screen approach that could employ genome-wide sgRNA libraries to systematically uncover genes crucial for these drugs action. Here we present our findings, including genes crucial for the import, export, metabolic activation and inactivation of remdesivir, as well as genes that regulate colchicine and dexamethasones immunosuppressive effects. Our findings provide preliminary information for developing urgently needed genetic biomarkers for these drugs. Such biomarkers will help better interpret COVID-19 clinical trial data and point to how to stratify COVID-19 patients for proper treatment with these drugs.

pharmacology and toxicology↗

bioRxiv2020

Transposable element expression at unique loci in single cells with CELLO-seq

The role of Transposable Elements (TEs) in regulating diverse biological processes, from early development to cancer, is becoming increasing appreciated. However, unlike other biological processes, next generation single-cell sequencing technologies are ill-suited for assaying TE expression: in particular, their highly repetitive nature means that short cDNA reads cannot be unambiguously mapped to a specific locus. Consequently, it is extremely challenging to understand the mechanisms by which TE expression is regulated and how they might themselves regulate other protein coding genes. To resolve this, we introduce CELLO-seq, a novel method and computational framework for performing long-read RNA sequencing at single cell resolution. CELLO-seq allows for full-length RNA sequencing and enables measurement of allelic, isoform and TE expression at unique loci. We use CELLO-seq to assess the widespread expression of TEs in 2-cell mouse blastomeres as well as human induced pluripotent stem cells (hiPSCs). Across both species, old and young TEs showed evidence of locus-specific expression, with simulations demonstrating that only a small number of very young elements in the mouse could not be mapped back to with high confidence. Exploring the relationship between the expression of individual elements and putative regulators revealed surprising heterogeneity, with TEs within a class showing different patterns of correlation, suggesting distinct regulatory mechanisms.

molecular biology↗