Biology subjects

Feng, H.

Publications and source records attributed to Feng, H..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Longitudinal profiling of B cells primed by mRNA vaccine and recalled by Omicron variants uncovers antibodies broadly neutralizing sarbecoviruses

A key question on ancestral SARS-CoV-2 immune imprinting is to what extent imprinted B cells can develop neutralizing breadth and potency in immune recalls. Here, we longitudinally tracked B cells recognizing wild-type spike in two individuals, who were sequentially infected by Omicron variants after receiving mRNA vaccines. Functional and genetic analysis of 632 monoclonal antibodies (mAbs) from those B cells reveals that mAbs cloned after second infection have dramatically enhanced neutralizing breadth and potency, which is attributed to recall and maturation of pre-existing memory B cells. Among the 11 mAbs that potently neutralize SARS-CoV-2 variants from wild-type to KP.3, 5 mAbs are classified into public clonotypes encoded by IGHV3-53 or IGHV3-66, whereas the rest belong to a rarely reported clonotype encoded by IGHV3-74. Notably, IGHV3-74 mAbs can also broadly neutralize other sarbecoviruses by targeting a novel epitope on receptor-binding domain of spike. These results support that ancestral SARS-CoV-2 immune imprinting can be harnessed in developing pan-SARS-CoV-2 and even pan-sarbecovirus vaccines. SummaryChen et al. demonstrate that B cells imprinted by ancestral SARS-CoV-2 have tremendous potential to develop neutralizing breadth and potency in repeated immune recalls driven by Omicron variants, implicating that ancestral SARS-CoV-2 immune imprinting can be harnessed in developing pan-SARS-CoV-2 and even pan-sarbecovirus vaccines.

immunology↗

bioRxiv2024

Benchmarking DNA Foundation Models for Genomic Sequence Classification

The rapid evolution of DNA foundation models promises to revolutionize genomics, yet comprehensive evaluations are lacking. Here, we present a comprehensive, unbiased benchmark of five models (DNABERT-2, Nucleotide Transformer V2, HyenaDNA, Caduceus-Ph, and GROVER) across diverse genomic and genetic tasks including sequence classification, gene expression prediction, variant effect quantification, and topologically associating domain (TAD) region recognition, using zero-shot embeddings. Our analysis reveals that mean token embedding consistently and significantly improves sequence classification performance, outperforming other pooling strategies. Model performance varies among tasks and datasets; while general purpose DNA foundation models showed competitive performance in pathogenic variant identification, they were less effective in predicting gene expression and identifying putative causal QTLs compared to specialized models. Our findings offer a framework for model selection, highlighting the impact of architecture, pre-training data, and embedding strategies on performance in genomic and genetic tasks.

genomics↗

bioRxiv2024

Coordinated regulation of chemotaxis and resistance to copper by CsoR in Pseudomonas putida

Copper is an essential enzyme cofactor in bacteria, but excess copper is highly toxic. Bacteria can cope with copper stress by increasing copper resistance and initiating chemorepellent response. However, it remains unclear how bacteria coordinate chemotaxis and resistance to copper. By screening proteins that interacted with the chemotaxis kinase CheA, we identified a copper-binding repressor CsoR that interacted with CheA in Pseudomonas putida. CsoR interacted with the HPT (P1), Dimer (P3), and HATPase_c (P4) domains of CheA and inhibited CheA autophosphorylation, resulting in decreased chemotaxis. The copper-binding of CsoR weakened its interaction with CheA, which relieved the inhibition of chemotaxis by CsoR. In addition, CsoR bound to the promoter of copper-resistance genes to inhibit gene expression, and copper-binding released CsoR from the promoter, leading to increased gene expression and copper resistance. P. putida cells exhibited a chemorepellent response to copper in a CheA-dependent manner, and CsoR inhibited the chemorepellent response to copper. Besides, the CheA-CsoR interaction also existed in proteins from several other bacterial species. Our results revealed a mechanism by which bacteria coordinately regulated chemotaxis and resistance to copper by CsoR.

microbiology↗

bioRxiv2024

PBRM1 directs PBAF to pericentromeres and protects centromere integrity

The specialised structure of the centromere is critical for effective chromosome segregation, but its repetitive nature makes it vulnerable to rearrangements. Centromere fragility can drive tumorigenesis, but protective mechanisms preventing fragility are still not fully understood. The PBAF chromatin remodelling complex is frequently misregulated in cancer, but its role in cancer is still not fully characterized. Here, we identify PBAF as a protector of centromere and pericentromere structure with profound consequences for genome stability. A conserved feature of isogenic cell lines lacking PBRM1, a subunit of PBAF, is compromised centromere and pericentromere integrity. PBAF is present at these regions, and the binding pattern changes when PBRM1 is absent. PBRM1 loss creates a dependence on the spindle assembly checkpoint, which represents a therapeutic vulnerability. Importantly, we find that even in the absence of any perturbations, PBRM1 loss leads to centromere fragility, thus identifying a new player in centromere protection.

molecular biology↗

bioRxiv2024

Reverse engineering neuron type-specific and type-orthogonal splicing-regulatory networks using single-cell transcriptomes

Cell type-specific alternative splicing (AS) enables differential gene isoform expression between diverse neuron types with distinct identities and functions. Current studies linking individual RNA-binding proteins (RBPs) to AS in a few neuron types underscore the need for holistic modeling. Here, we use network reverse engineering to derive a map of the neuron type-specific AS regulatory landscape from 133 mouse neocortical cell types defined by single-cell transcriptomes. This approach reliably inferred the regulons of 350 RBPs and their cell type-specific activities. Our analysis revealed driving factors delineating neuronal identities, among which we validated Elavl2 as a key RBP for MGE-specific splicing in GABAergic interneurons using an in vitro ESC differentiation system. We also identified a module of exons and candidate regulators specific for long- and short-projection neurons across multiple neuronal classes. This study provides a resource for elucidating splicing regulatory programs that drive neuronal molecular diversity, including those that do not align with gene expression-based classifications.

systems biology↗

bioRxiv2024

Precise cell recovery by cell nucleus united transcript (CellCUT) for enhanced spatial transcriptomics

Cell segmentation is the first step in parsing spatial transcriptomic data, often a challenging task. Existing cell segmentation methods do not fully leverage spatial cues between nuclear images and transcripts, tending to produce undesirable cell profiles for densely packed cells. Here, we propose CellCUT to perform cell segmentation and transcript assignment without additional manual annotations. CellCUT provides a flexible computational framework that maintains high segmentation accuracy across diverse tissues and spatial transcriptomics protocols, showing superior capabilities compared to state-of-the-art methods. CellCUT is a robust model to deal with undesirable data such as low contrast intensity, localized absence of transcripts, and blurred images. CellCUT supports a human-in-the-loop workflow to enhance its generalizability to customized datasets. CellCUT identifies subcellular structures, enabling insights at both the single-cell and subcellular levels.

bioinformatics↗

bioRxiv2024

Ancestral SARS-CoV-2 immune imprinting persists on RBD but not NTD after sequential Omicron infections

Whether Omicron exposures could overcome ancestral SARS-CoV-2 immune imprinting remains controversial. Here we analyzed B cell responses evoked by sequential Omicron infections in vaccinated and unvaccinated individuals. Plasma neutralizing antibody titers against ancestral SARS-CoV-2 and variants indicate that immune imprinting is not consistently induced by inactivated or recombinant protein vaccines. However, once induced, immune imprinting is not countered by successive Omicron challenges. We compared binding specificities, neutralizing capacities, developing origins and targeting epitopes of monoclonal antibodies from individuals with or without immune imprinting. Although receptor-binding domain (RBD) and N-terminal domain (NTD) of spike are both primary targets for neutralizing antibodies, immune imprinting only shapes antibody responses to RBD by impeding the production of Omicron-specific neutralizing antibodies while facilitating the development of broadly neutralizing antibodies. We propose that immune imprinting can be either neglected by NTD-based vaccines to induce variant-specific antibodies or leveraged by RBD-based vaccines to induce broadly neutralizing antibodies.

immunology↗

bioRxiv2024

Natural selection and evolution of mitochondrial genome in terrestrial and aquatic turbellarians

Mitochondrial energy metabolism may be directly influenced by natural selection pressures in response to the energy demands of an organisms specific ecological niche. Here, we hypothesized that the mitochondrial genome of turbellarian animals was influenced by the metabolic requirements of various habitats We used selection pressure and phylogenetic independent contrasts (PIC) analyses to detect the selection pressure of protein-coding genes in the mitochondrial genome of turbellarian living in aquatic and terrestrial habitats. The results indicated turbellarians in aquatic habitats experienced greater selection pressure, resulting in the evolution of several genes at a lower {omega} value compared to their terrestrial counterparts. The NAD4 was identified as having positive selection sites in all site models analyzed. Among all genes, the equilibrium constant (ionization of COOH) was the most frequently detected amino acid characteristic with significant positive selection changes, followed by hydropathy and molecular volume. The equilibrium constant (ionization of COOH) was found to be the amino acid characteristic with the greatest magnitude of change in NAD4. This suggests that it plays a crucial role in the adaptive evolution of turbellarians to varying habitats. The study found a significant positive correlation (R = 0.61, p < 0.05) between the {omega} value of turbellarian and their habitats by PIC analysis. These findings shed light on the adaptive evolution of turbellarian mtDNAs and their influence on the oxidative phosphorylation molecular mechanism. 0. Significance statementThe adaptive evolution of mitochondrial genes is closely related to environmental temperature, high energy metabolism demand, altitude, and oxygen availability. Mitochondrial energy metabolism may be directly influenced by natural selection pressures in response to the energy demands of an organisms specific ecological niche. Here, we investigated the evolution of mtDNA PCGs in turbellarians across different habitats, and identified purifying selection as the main evolutionary pattern of the turbellarian mitochondrial genome. Additionally, we found that NAD4 played an important role in the adaptive evolution of turbellarians to different habitats.

evolutionary biology↗

bioRxiv2024

PFKP regulates AXL-MET oncogenic and metabolic pathways in lung cancer

ObjectivePFKP (Phosphofructokinase, Platelet Type isoform), as an essential metabolic enzyme, contributes to the high glycolysis rates seen in cancers, while its role in oncogenic pathways, especially from a non-metabolic aspect, is not fully understood. MethodsHere we performed a comprehensive analysis of published RNA-seq, microarray data, and immunohistochemistry of tissue microarray to evaluate the significance of PFKP expression in non-small cell lung cancer (NSCLC). Functionally, we tested the cell proliferation, colony formation, invasion, and migration upon PFKP knockdown in lung cancer cells. Mechanistically, we performed RNA-seq, DIA-mass spectrum, western blot, and qPCR to probe the change of cell signaling pathways upon PFKP silencing. Co-immunoprecipitation and mass spectrum were used to uncover potential PFKP interacting proteins. ResultsWe found that PFKP was highly expressed in NSCLC and was related to poor patient survival. Knockdown of PFKP significantly inhibited cell proliferation, colony formation, invasion, and migration of NSCLC cells. Mechanistically, we found that PFKP can directly bind with AXL and promote its phosphorylation at Y779, thus activating the AXL signaling pathway and promoting MET phosphorylation. In addition, several glycolysis, glutaminolysis, and TCA cycle proteins were downregulated following PFKP silencing. ConclusionsThese data demonstrate that PFKP, beyond its known role in glycolysis, also has a distinct non-metabolic function in affecting lung cancer progression by directly interacting with the AXL-MET axis, thus indicating a potential therapeutic target for lung cancer.

cancer biology↗

bioRxiv2024

Systematic Identification of UBE2C As a Prognostic Biomarker and Correlated with Immunosuppression and Invasiveness in Glioma

Glioma is one of the common tumors of the central nervous system, which presents difficulties in clinical diagnosis and treatment due to its characteristics of immunosuppression and cell invasion phenotypes. If the condition and prognosis of glioma can be predicted during the process of diagnosis and treatment, it will be more conducive to timely intervention or evaluation of glioma. Therefore, we still need to search for more valuable tumor markers. The differential/risk genes and enrichment analysis based on glioma samples (The Cancer Genome Atlas, TCGA). Target gene UBE2C were obtained by the expression correlation and differential expression analysis for the enrichment results. UBE2C were evaluated by clinical grading, survival prognosis and cell experiments. The correlation of UBE2C with immune invasion, immune checkpoint, network analysis and cell invasiveness of gliomas was analyzed by TCGA-glioma data and STRING, respectively. The results suggests that the high expression and risk of UBE2C in gliomas may be a factor that promotes malignant phenotype of tumor cells. The immune phenotype shows that IL6 and IL10 may be the key nodes affecting the immunosuppressive phenotype of glioma. Further, the tumor cells aggressive genes from the MMP family can be correlated with immunosuppressive phenotypes via UBE2C-IL6/IL10 axis, especially displayed by MMP2/MMP9. The UBE2C may systemic effects the malignant phenotype, immunosuppression and cell invasiveness of tumors systematically, which reflects UBE2C as a potential biomarker of glioma and therapeutic target for this tumor.

cancer biology↗

bioRxiv2024

Pooled CRISPR interference screening identifies crucial transcription factors in gas-fermenting Clostridium ljungdahlii

Gas-fermenting Clostridium species hold tremendous promise for one-carbon biomanufacturing. To unlock their full potential, it is crucial to unravel and optimize the intricate regulatory networks that govern these organisms; however, this aspect is currently underexplored. In this study, we employed pooled CRISPR interference (CRISPRi) screening to uncover a wide range of functional transcription factors (TFs) in Clostridium ljungdahlii, a representative species of gas-fermenting Clostridium, with a special focus on the TFs associated with the utilization of carbon resources. Among the 425 TF candidates, we identified 75 and 68 TF genes affecting the heterotrophic and autotrophic growth of C. ljungdahlii, respectively. We directed our attention on two of the screened TFs, NrdR and DeoR, and revealed their pivotal roles in the regulation of deoxyribonucleotides (dNTPs) supply, carbon fixation, and product synthesis in C. ljungdahlii, thereby influencing the strain performance in gas fermentation. Based on this, we proceeded to optimize the expression of deoR in C. ljungdahlii by adjusting its promoter strength, leading to improved growth rate and ethanol synthesis of C. ljungdahlii when utilizing syngas. This study highlights the effectiveness of pooled CRISPRi screening in gas-fermenting Clostridium species, expanding the horizons for functional genomic research in these industrially important bacteria.

microbiology↗

bioRxiv2024

Nephrectomy and high-salt diet inducing pulmonary hypertension and kidney damage by increasing Ang II concentration in rats

BackgroundPulmonary hypertension (PH) is a common complication in patients with chronic kidney disease (CKD), affecting prognosis. However, the pathogenesis is not clear, and the lack of a stable animal model is a significant factor. MethodsIn this study, a rat model of chronic kidney disease with pulmonary hypertension (CKD-PH) was developed through 5/6 nephrectomy combined with a high-salt diet. The models hemodynamics and pathological changes in multiple organs were dynamically assessed. Lung tissues and serum were collected from the model rats to measure the expression of ACE2, the expression levels of vascular active components related to the renin-angiotensin-aldosterone system (RAAS), and changes in the serum metabolic profile of the model. ResultsAfter 14 weeks post-surgery, the CKD-PH rat model exhibited significant changes in hemodynamic parameters indicative of pulmonary arterial hypertension, along with alterations such as right ventricular hypertrophy. However, no evidence of pulmonary vascular remodeling was observed. An imbalance in the renin-angiotensin-aldosterone system was identified in the CKD-PH rat models. Downregulation of ACE2 expression was observed in pulmonary tissues. The serum metabolic profile of the CKD-PH rat models showed distinct differences compared to the sham surgery group. ConclusionsThe development of pulmonary arterial hypertension in CKD-PH rats may be primarily attributed to the disruption of the renin-angiotensin-aldosterone system (RAAS), coupled with a decrease in ACE2 expression in pulmonary vascular endothelial tissues and metabolic disturbances.

pathology↗

bioRxiv2024

Genome and tissue-specific transcriptome of the tropical milkweed (Asclepias curassavica)

Tropical milkweed (Asclepias curassavica) serves as a host plant for monarch butterflies (Danaus plexippus) and other insect herbivores that can tolerate the abundant cardiac glycosides that are characteristic of this species. Cardiac glycosides, along with additional specialized metabolites, also contribute to the ethnobotanical uses of A. curassavica. To facilitate further research on milkweed metabolism, we assembled the 197 Mbp genome of a fifth-generation inbred line of A. curassavica into 619 contigs, with an N50 of 10 Mbp. Scaffolding resulted in 98% of the assembly being anchored to 11 chromosomes, which are mostly colinear with the previously assembled common milkweed (A. syriaca) genome. Assembly completeness evaluations showed that 98% of the BUSCO gene set is present in the A. curassavica genome assembly. The transcriptomes of six tissue types (young leaves, mature leaves, stems, flowers, buds, and roots), with and without defense elicitation by methyl jasmonate treatment, showed both tissue-specific gene expression and induced expression of genes that may be involved in cardiac glycoside biosynthesis. Expression of a CYP87A gene, the predicted first gene in the cardiac glycoside biosynthesis pathway, was observed only in the stems and roots and was induced by methyl jasmonate. Together, this genome sequence and transcriptome analysis provide important resources for further investigation of the ecological and medicinal uses of A. curassavica.

plant biology↗

bioRxiv2023

Integrated Transcriptomic and Metabolomic Profiling of Sheep Ovarian Tissues Confer their association in Fecundity associated Pathways

BackgroundLow fertility is considered the major constraint in sheep rearing industry depending on several factors like, estrus cycle, ovulation rate and litter size but fecundity of ewe plays a key role in sheep reproduction, influenced by several intrinsic and extrinsic factors. However, genetic improvements of traits associated with reproduction through conventional breeding is a very complex and slow process. In current study, we went through a comprehensive integration of high throughput transcriptomic and metabolomics approaches to understand the role of key regulatory genes and metabolites in fecundity of two different and widely raised sheep breeds (Small Tail Han & Dolang) in different regions of China. ResultUPLC/MS/MS system based metabolomic profiling of ovarian tissue from both breeds results into the identification of 1,423 metabolites, including 542 DEMs (379 upregulated and 163 downregulated). Integration of metabolomics and transcriptomics data identified 48 pathways contributed by 37 genes and 85 metabolites through regulatory network analysis. Functional enrichment analysis showed significantly enriched pathways associated with fecundity including Riboflavin metabolism, xenobiotics, bile acid biosynthesis, and Drug metabolism, which produces hormones for regulation of ovarian function, ovulation, and establishment of pregnancy. Further, analyzed two restrictive constrained plots analyzed via multivariate statistical analysis. In one plot complement component C3 associated with Leukotriene D4, and Uridine 5-diphosphate involved in the processes of Neuroactive legend receptor interaction pathway and in second plot IFNGR1 associated with Progesterone, Fumaric acid, and Cortisone involved in the processes of cancer pathway and any disruptions in hormonal balance may induce cancer, which can affect fertility, menstrual cycles, and overall reproductive health. ConclusionExpression profiling, functional enrichments, co-expression network analysis and integrated transcriptomemetabolome data showed gene-metabolite association in energy metabolism, Inflammation, and drug metabolism, all of which play a role in ovarian physiology and ovarian metabolic disorders. Identification and validation of genes, metabolites, and gene-metabolite interactions will help to elucidate the regulatory mechanisms and pathways underlying sheep fecundity and could be leveraged to improve reproductive traits. Graphical Abstract: Scheme of Study O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/572136v1_ufig1.gif" ALT="Figure 1"> View larger version (119K): org.highwire.dtl.DTLVardef@12a42b7org.highwire.dtl.DTLVardef@182169forg.highwire.dtl.DTLVardef@19b2ed4org.highwire.dtl.DTLVardef@11f9eab_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Integrated Hfq-interacting RNAome and transcriptomic analysis reveals complex regulatory networks of nitrogen fixation in root-associated Pseudomonas stutzeri A1501

The RNA chaperone Hfq acts as a global regulator of numerous biological processes, such as carbon/nitrogen metabolism and environmental adaptation in plant-associated diazotrophs; however, its target RNAs and the mechanisms underlying nitrogen fixation remain largely unknown. Here, we used enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing (eCLIP-seq) to identify hundreds of Hfq-binding RNAs probably involved in nitrogen fixation, carbon substrate utilization, biofilm formation, and other functions. Collectively, these processes endow strain A1501 with the requisite capabilities to thrive in the highly competitive rhizosphere. Our findings revealed a previously uncharted landscape of Hfq target genes. Notable among these is nifM, encoding an isomerase necessary for nitrogenase reductase solubility; amtB, encoding an ammonium transporter; oprB, encoding a carbohydrate porin; and cheZ, encoding a chemotaxis protein. Furthermore, we identified more than one hundred genes of unknown function, which expands the potential direct regulatory targets of Hfq in diazotrophs. Our data showed that Hfq directly interacts with regulatory proteins (RsmA, AlgU, NifA), regulatory ncRNA RsmY, and other potential targets, thus revealing the mechanistic links in nitrogen fixation and other metabolic pathways. IMPORTANCENumerous experimental approaches often face challenges in distinguishing between direct and indirect effects of Hfq-mediated regulation. New technologies based on high-throughput sequencing are increasingly providing insight into the global regulation of Hfq in gene expression. Here, enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing (eCLIP-seq) was employed to identify the Hfq-binding sites and potential targets in the root-associated P. stutzeri A1501, and identify hundreds of novel Hfq-binding RNAs that are predicted to be involved in metabolism, environmental adaptation, and nitrogen fixation. In particular, we have shown that Hfq interactions with various regulatory proteins and their potential targets at both the protein and RNA levels. This study not only enhances our understanding of Hfq regulation but, importantly, also provides a framework for addressing integrated regulatory network underlying root-associated nitrogen fixation.

microbiology↗

bioRxiv2023

imply: improving cell-type deconvolution accuracy using personalized reference profiles

Real-world clinical samples are often admixtures of signal mosaics from multiple pure cell types. Using computational tools, bulk transcriptomics can be deconvoluted to solve for the abundance of constituent cell types. However, existing deconvolution methods are conditioned on the assumption that the whole study population is served by a single reference panel, which ignores person-to-person heterogeneity. Here we present imply, a novel algorithm to deconvolute cell type proportions using personalized reference panels. imply can borrow information across repeatedly measured samples for each subject, and obtain precise cell type proportion estimations. Simulation studies demonstrate reduced bias in cell type abundance estimation compared with existing methods. Real data analyses on large longitudinal consortia show more realistic deconvolution results that align with biological facts. Our results suggest that disparities in cell type proportions are associated with several disease phenotypes in type 1 diabetes and Parkin-sons disease. Our proposed tool imply is available through the R/Bioconductor package ISLET at https://bioconductor.org/packages/ISLET/.

bioinformatics↗

bioRxiv2023

magpie: a power evaluation method for differential RNA methylation analysis in N6-methyladenosine sequencing

MotivationRecently, novel biotechnologies to quantify RNA modifications became an increasingly popular choice for researchers who study epitran-scriptome. When studying RNA methylations such as N6-methyladenosine (m6A), researchers need to make several decisions in its experimental design, especially the sample size and a proper statistical power. Due to the complexity and high-throughput nature of m6A sequencing measurements, methods for power calculation and study design are still currently unavailable. ResultsWe propose a statistical power assessment tool, magpie, for power calculation and experimental design for epitranscriptome studies using m6A sequencing data. Our simulation-based power assessment tool will borrow information from real pilot data, and inspect various influential factors including sample size, sequencing depth, effect size, and basal expression ranges. We integrate two modules in magpie: (i) a flexible and realistic simulator module to synthesize m6A sequencing data based on real data; and (ii) a power assessment module to examine a set of comprehensive evaluation metrics. AvailabilityThe proposed power assessment tool magpie is publicly available as a R/Bioconductor package at: https://bioconductor.org/packages/magpie/.

bioinformatics↗

bioRxiv2023

A single cell transcriptomic fingerprint of stressed premature, imbalanced differentiation of embryonic stem cells.

Cultured naive pluripotent ESC differentiate into first lineage, XEN or second lineage, formative pluripotency. Hyperosmotic stress (sorbitol), like retinoic acid, decreases naive pluripotency and increases XEN in two ESC lines, as reported by bulk and scRNAseq, analyzed by UMAP. Sorbitol overrides pluripotency in two ESC lines as reported by bulk and scRNAseq, analyzed by UMAP. UMAP analyzed the effects of 5 stimuli - three stressed (200-300mM sorbitol with leukemia inhibitory factor +LIF) and two unstressed (+LIF, normal stemness-NS and -LIF, normal differentiation-ND). Sorbitol and RA decrease naive pluripotency and increase subpopulations of 2-cell embryo-like and XEN sub-lineages; primitive, parietal, and visceral endoderm (VE). Between the naive pluripotency and primitive endoderm clusters is a stress-induced cluster with transient intermediate cells with higher LIF receptor signaling, with increased Stat3, Klf4, and Tbx3 expression. Sorbitol, like RA, also suppresses formative pluripotency, increasing lineage imbalance. Although bulk RNAseq and gene ontology group analyses suggest that stress induces head organizer and placental markers, scRNAseq reveals few cells. But VE and placental markers/cells were in adjacent clusters, like recent reports. UMAPs show that dose-dependent stress overrides stemness to force premature lineage imbalance. Hyperosmotic stress induces lineage imbalance, and other toxicological stresses, like drugs with RA, may cause lineage imbalance, resulting in miscarriages or birth defects.

developmental biology↗