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PubMed2025

Cell-type-specific dysregulation of gene expression due to Chd8 haploinsufficiency during mouse cortical development.

Disruptive variants in the chromodomain helicase CHD8 are associated with risk for autism spectrum disorder (ASD). CHD8 haploinsufficiency is hypothesized to contribute to ASD by perturbing neurodevelopmental gene expression. However, insight into cell-type-specific transcriptional effects of CHD8 haploinsufficiency remains limited. We used single-cell and single-nucleus RNA sequencing to identify dysregulated genes in the embryonic and juvenile Chd8+/- mouse cortex. Chd8 and other ASD risk-associated genes showed a convergent expression trajectory conserved between mouse and human developing cortex, increasing from progenitor zones to the cortical plate. Genes associated with neurodevelopmental disorders or involved in chromatin remodeling and neuron projection development were dysregulated in Chd8+/- embryonic radial glia. Genes implicated in synaptic activity and organization were dysregulated in Chd8+/- postnatal excitatory cortical neurons, suggesting impaired synaptogenesis. Our findings reveal complex patterns of transcriptional dysregulation due to Chd8 haploinsufficiency, potentially with distinct impacts on progenitors and maturing neurons in the excitatory neuronal lineage.

Animals↗
PubMed2021

Dissecting spatial heterogeneity and the immune-evasion mechanism of CTCs by single-cell RNA-seq in hepatocellular carcinoma.

Little is known about the transcriptomic plasticity and adaptive mechanisms of circulating tumor cells (CTCs) during hematogeneous dissemination. Here we interrogate the transcriptome of 113 single CTCs from 4 different vascular sites, including hepatic vein (HV), peripheral artery (PA), peripheral vein (PV) and portal vein (PoV) using single-cell full-length RNA sequencing in hepatocellular carcinoma (HCC) patients. We reveal that the transcriptional dynamics of CTCs were associated with stress response, cell cycle and immune-evasion signaling during hematogeneous transportation. Besides, we identify chemokine CCL5 as an important mediator for CTC immune evasion. Mechanistically, overexpression of CCL5 in CTCs is transcriptionally regulated by p38-MAX signaling, which recruites regulatory T cells (Tregs) to facilitate immune escape and metastatic seeding of CTCs. Collectively, our results reveal a previously unappreciated spatial heterogeneity and an immune-escape mechanism of CTC, which may aid in designing new anti-metastasis therapeutic strategies in HCC.

Aged↗
PubMed2025

Ovarian development is driven by early spatiotemporal priming of the coelomic epithelium.

Ovarian organogenesis requires the coordinated specification of supporting and steroidogenic cell lineages from multipotent coelomic epithelium (CE) progenitors. A longstanding question is whether the CE contains transcriptionally distinct, spatially organized progenitor subpopulations with predetermined lineage biases, or whether specification into supporting and steroidogenic lineages occurs only after delamination and integration into the bipotential gonad. The developmental origins of granulosa cells and the emergence of ovarian steroidogenic/stromal progenitors (SPs) also remain poorly defined. Here, we show that CE cells covering the fetal mouse ovary are transcriptionally heterogeneous and spatially organized into subdomains already primed toward supporting or steroidogenic fates. CE priming is dynamic, with transient coexistence of supporting- and steroidogenic-biased CE progenitors before resolving into a predominantly supporting-biased CE. Local delamination of these primed cells seeds intragonadal niches where pre-granulosa cells and SPs mirror the spatio-temporal arrangements of CE-primed progenitors. We further demonstrate a dual origin for the supporting lineage, with granulosa cells deriving from both the CE and supporting-like cells (SLCs). In parallel, we show that SPs arise from steroidogenic-primed CE cells, expand to represent 52% of ovarian somatic cells at birth, persist into adulthood and contribute to both theca and steroidogenic stromal cells. Together, these findings reveal transcriptionally and spatially distinct CE subpopulations that shape somatic lineage emergence with important implications for ovarian pathophysiology.

Ovarian development↗
PubMed2026

Follicular Lymphoma Transformation is Characterized by Cytokine-associated Remodeling of Stromal and Macrophage Compartments.

Across cancer, one of the most frequent examples of histologic transformation is the evolution of follicular lymphoma (FL) to an aggressive large cell lymphoma. Despite recent progress, understanding of the molecular and cellular underpinnings of transformation remains incomplete. Here, we dissect the interplay of tumor and microenvironment cell populations across transformation through a multimodal investigation of 95 FL and transformed FL (tFL) samples, including single-cell and bulk RNA-sequencing alongside spatial transcriptomics and proteomics, and validate findings across independent FL-tFL pairs. Upon transformation, fibroblasts and GPNMB+ macrophages increase while lymph-node organizing follicular dendritic and CCL21+ fibroblastic reticular cells were lost, resulting in an altered spatial distribution of cytokines that impacts T cell infiltration and macrophage differentiation and function. Secreted stromal and macrophage signals were further evident by non-invasive plasma proteomics. Taken together, our data reveal expansion of macrophages and fibroblasts as key features of transformation with potential diagnostic and therapeutic implications.

Journal Article↗
PubMed2026

Machine learning-integrated multi-omics risk prediction for pulmonary fungal infection in COPD and lung cancer: a transcriptomic and immune profiling study.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) and lung cancer are major risk factors for invasive pulmonary fungal infection (IPFI), carrying an attributable mortality of 30%-80%. Their coexistence further amplifies immunosuppression, while current diagnostic criteria remain inadequate for early risk identification. METHODS: Transcriptomic data from the GEO dataset GSE296912 (scRNA-seq; 12,078 cells from normal and COPD lung tissue) and The Cancer Genome Atlas (TCGA)-lung adenocarcinoma (LUAD) bulk RNA-seq cohort (539 tumor and 59 normal samples) underwent differential expression and cross-omics integration analysis. Five machine learning models were constructed: logistic regression, SVM, random forest, XGBoost, and LASSO. Candidate genes were validated by qRT-PCR in A549 cells and THP-1-derived macrophages stimulated with heat-inactivated Aspergillus fumigatus conidia, a protocol selected to ensure BSL-2 biosafety compliance and isolate PAMP-mediated innate immune signaling. Model performance was evaluated using 5-fold stratified cross-validation with AUC, calibration curves, and decision curve analysis. RESULTS: Single-cell transcriptomic analysis of 12,078 cells identified 14 distinct cell populations, with marked myeloid expansion and immune dysregulation in COPD lung tissue. Cross-omics integration with TCGA-LUAD data identified 1,145 shared genes (79 immune-related), converging on NF-κB, TLR4, and cytokine receptor signaling. The random forest model achieved excellent discriminative performance (5-fold CV AUC = 0.988), with Treg infiltration, TLR4, and MMP9 as the top predictors. qRT-PCR confirmed significant upregulation of all five candidate genes (DEFB4A, S100A8, IL-8, MMP9, and TLR4) in both A549 and THP-1 cells following fungal stimulation. CONCLUSION: This multi-omics machine learning model integrating scRNA-seq and TCGA transcriptomic data demonstrates excellent discriminative performance (AUC = 0.988), with mechanistic convergence of NF-κB, TLR4, and oncogenic signaling pathways identified across shared immune gene signatures. In vitro qRT-PCR validation confirms the biological relevance of five key antifungal immune genes, providing a transcriptomic foundation for future prospective IPFI risk stratification in patients with COPD and lung cancer.

TLR4↗
PubMed2025

Molecular hallmarks of excitatory and inhibitory neuronal resilience to Alzheimer's disease.

BACKGROUND: A significant proportion of individuals maintain cognition despite extensive Alzheimer's disease (AD) pathology, known as cognitive resilience. Understanding the molecular mechanisms that protect these individuals could reveal therapeutic targets for AD. METHODS: This study defines molecular and cellular signatures of cognitive resilience by integrating bulk RNA and single-cell transcriptomic data with genetics across multiple brain regions. We analyzed data from the Religious Order Study and the Rush Memory and Aging Project (ROSMAP), including bulk RNA sequencing (n = 631 individuals) and multiregional single-nucleus RNA sequencing (n = 48 individuals). Subjects were categorized into AD, resilient, and control based on β-amyloid and tau pathology, and cognitive status. We identified and prioritized protected cell populations using whole-genome sequencing-derived genetic variants, transcriptomic profiling, and cellular composition. RESULTS: Transcriptomics and polygenic risk analysis position resilience as an intermediate AD state. Only GFAP and KLF4 expression distinguished resilience from controls at tissue level, whereas differential expression of genes involved in nucleic acid metabolism and signaling differentiated AD and resilient brains. At the cellular level, resilience was characterized by broad downregulation of LINGO1 expression and reorganization of chaperone pathways, specifically downregulation of Hsp90 and upregulation of Hsp40, Hsp70, and Hsp110 families in excitatory neurons. MEF2C, ATP8B1, and RELN emerged as key markers of resilient neurons. Excitatory neuronal subtypes in the entorhinal cortex (ATP8B+ and MEF2Chigh) exhibited unique resilience signaling through activation of neurotrophin (BDNF-NTRK2, modulated by LINGO1) and angiopoietin (ANGPT2-TEK) pathways. MEF2C+ inhibitory neurons were over-represented in resilient brains, and the expression of genes associated with rare genetic variants revealed vulnerable somatostatin (SST) cortical interneurons that survive in AD resilience. The maintenance of excitatory-inhibitory balance emerges as a key characteristic of resilience. CONCLUSIONS: We have defined molecular and cellular hallmarks of cognitive resilience, an intermediate state in the AD continuum. Resilience mechanisms include preserved neuronal function, balanced network activity, and activation of neurotrophic survival signaling. Specific excitatory neuronal populations appear to play a central role in mediating cognitive resilience, while a subset of vulnerable interneurons likely provides compensation against AD-associated hyperexcitability. This study offers a framework to leverage natural protective mechanisms to mitigate neurodegeneration and preserve cognition in AD.

Humans↗
PubMed1993

A simple nucleic acid amplification assay for the rapid detection of Junín virus in whole blood samples.

Argentine hemorrhagic fever (AHF) is an endemoepidemic disease with cardiovascular, renal and neurologic alterations acquired in the richest farming land in Argentina. It is caused by Junín virus, one of the few human pathogenic arenaviruses. The S RNA of Junín virus has been molecularly cloned and its nucleotide sequence determined in our laboratory. This information was used to develop a rapid nucleic acid-based diagnostic test commensurate with the low viraemia detected in AHF patients. Junín virus-specific cDNA probes labeled using various methods proved insensitive in dot-hybridizations. Therefore, a RT polymerase chain reaction (PCR) was developed using a pair of oligonucleotide primers to reverse-transcribe and amplify the viral S RNA. The amplification of the target sequences was measured by ethidium bromide staining of the DNA fragments after agarose gel electrophoresis. This type of assay allowed the specific detection of Junín virus RNA sequences present in a single infected BHK21 cell over a background of 10(4) uninfected cells. Control reactions were performed on RNA samples extracted from uninfected cells or cells infected with a high multiplicity of LCMV, another arenavirus present in the AHF endemic area. The PCR was first adapted to detect viral RNA in peripheral blood mononuclear cells, described to harbor most of the virus. A simplification of this assay allows the detection of Junín virus in RNA extracted from 100 microliters of whole blood using guanidium thiocyanate disruption and acid phenol extraction. Under the conditions described in this paper, it is possible to detect up to 0.01 pfu of Junín virus in a blood sample. An early and rapid laboratory diagnostic test for AHF is important since the only effective therapy that reduces the mortality rate from 30% to less than 1% consists of early treatment with immune plasma.

Animals↗
PubMed2026

Precision-Based Filtering Facilitates Cross-Referencing of Conventional and Single-Nucleus Transcriptomes to Identify Time- and Temperature-Sensitive Cell Populations.

Transcriptome analysis via RNA sequencing (RNAseq) has become a ubiquitous method of molecular characterization from whole organisms, dissected tissues, and single cells. These experiments continue to provide an extraordinary volume of data describing molecular states and responses to many conditions. However, standard approaches to RNAseq analysis commonly use expression level filters that eliminate potentially useful data in the service of decreasing noise. Here we describe the implementation of a coefficient of variation-based filter for RNAseq gene expression data. This filter prioritizes consistent data across replicates, allowing lowly-expressed genes with low-variation measurements to be retained for downstream analysis. We show, using two independent Arabidopsis RNAseq datasets, that this filter allows for the inclusion of many more transcription factors than even a low-stringency expression level filter. This effect is independent of sequencing depth. We find that these lowly-expressed genes mark specific cell clusters in our single-nucleus (sn)RNAseq dataset and may facilitate future characterization of currently unknown cell types or states. We further characterize communities of co-expressed genes, sampled across the day at two growth temperatures, in relation to snRNAseq cell clusters, finding evidence for a highly photosynthetic cell population, and a cell state marked by high cell division and translation. These methods can be expanded to RNAseq analysis in many systems, facilitating the construction of more detailed models of tissue-specific gene regulatory networks.

Transcriptome analysis↗
PubMed1978

Bovine leukemia virus (BLV) specific RNA in infected cells.

BLV specific RNA sequences were detected by hybridization to the single stranded DNA copy of the viral RNA in fetal lamb kidney cells infected with bovine leukemia virus (FLK-BLV). The DNA synthesis was carried out in vitro by an endogenous reaction. It was found that uninfected lamb cells do not contain RNA sequences related to the BLV genome, whereas BLV-DNA completely annealed to FLK-BLV RNAs. RNA from FLK-BLV cells contains three species of virus specific RNA which sediment respectively at 35 S, 20--24 S and 12--4 S. In view of their EDTA-sensitive association with ribosomes, viral specific RNA sequences are viral messenger RNAs and their polarity is identical to that of the 70 S viral RNA.

Cells, Cultured↗
PubMed1999

Involvement of cis-acting repressive element(s) in the 3'-untranslated region of human connective tissue growth factor gene.

To analyze the regulatory mechanism of connective tissue growth factor expression, the 3'-untranslated region (3'-UTR) of CTGF cDNA was amplified from HeLa cell RNA. Direct nucleotide sequencing revealed a single major population in the amplicon, which was nearly identical to other sequences. Subsequently, the effect of the 3'-UTR on gene expression was evaluated. When it was fused downstream of a firefly luciferase gene, the 3'-UTR strongly repressed luciferase gene expression. Interestingly, the repressive effect of the antisense 3'-UTR appeared to be more prominent than that of the sense one. Together with the fact that several consensus sequences for regulatory elements are found in it, these results suggest the involvement of multiple sets of regulatory elements in the CTGF 3'-UTR.

3' Untranslated Regions↗
PubMed1992

Vegetal messenger RNA localization directed by a 340-nt RNA sequence element in Xenopus oocytes.

Contained within a single cell, the fertilized egg, is information that will ultimately specify the entire organism. During early embryonic cleavages, cells acquire distinct fates and their differences in developmental potential might be explained by localization of informational molecules in the egg. The mechanisms by which Vg1 RNA, a maternal mRNA, is translocated to the vegetal pole of Xenopus oocytes may indicate how developmental signals are localized. Data presented here show that a 340-nucleotide localization signal present in the 3' untranslated region of Vg1 RNA is sufficient to direct RNA localization to the vegetal pole.

Animals↗
PubMed2025

The offonome reveals on and off states of gene expression near the detection limit of RNA-seq.

RNA-seq, widely used for gene expression profiling, provides nucleotide level genome coverage and summary gene expression values. Generally, low-expressed genes are ignored due to their unfavorable signal-to-noise ratio, however, these genes may offer crucial information, such as detecting rare cells in bulk tissues. In this study, we applied an approach that transforms the expression levels of low-expressed genes into a robust dichotomized on/off state by leveraging similarities in transcript coverage shape. Applied to three human cancer cohorts from the Cancer Genome Atlas (TCGA), chosen based on tissue morphology and anatomic site, we identified genes, the "offonome" near the detection limit, consistently or occasionally off across samples. Genes in the offonome spectrum proved useful for supervised and unsupervised applications, including characterizing oncogenic pathways, and identifying rare populations of cells in bulk tissue. Interrogating the offonome is relevant to bulk tumor analyses like TCGA, potentially expediting gene investigation in low-input situations like single cell RNA-seq.

Humans↗
PubMed1974

Interspersion of repetitive and single-copy sequences in nuclear ribonucleic acid of high molecular weight.

Nuclear RNA of high molecular weight, approximately 15 to 30 x 10(3) nucleotides long, isolated from rat ascites cells, has been shown previously to contain both middle repetitive and single-copy sequences. However, it was not known whether these sequences were located on separate molecules or whether they were interspersed within the same molecule. The present communication distinguishes between these possibilities and shows that a large proportion of nuclear RNA of high molecular weight consists of interspersed middle repetitive and single-copy sequences.

Animals↗
PubMed1988

Comparative analysis of polyadenylated RNA complexity in soybean hypocotyl tissue and cultured suspension cells.

Growth parameters of suspension culture cells of soybean (Glycine max L.) were compared between cells grown in medium with (+) auxin and without (-) auxin. Growth rates were greater for (+) auxin cells. Cells transferred to (-) auxin medium primarily expanded in size while (+) auxin cells initially divided and then expanded. Two methods were used to estimate polyadenylated RNA sequence complexity. Kinetic analysis gave a sum of component complexity values of 36,000 and 64,000 diverse poly(A) RNA sequences of about 1,400 nucleotides in (+) and (-) auxin grown cells, respectively. The most striking difference between these cell populations was the increase in the poly(A) RNA sequence complexity in cells grown in medium without auxin. RNA complexities were also determined by the saturation of ;single' copy DNA by poly(A) RNAs from (+) and (-) auxin suspension cells. These saturation studies estimated the total complexity of (+) and (-) auxin suspension cells as 41,000 and 57,000 diverse sequences, respectively. Suspension cells in auxin-depleted medium produced about 20,000 more diverse sequences than (+) auxin cells. Comparisons of poly(A) complexities were also made among auxin-treated and untreated hypocotyl cells from the intact plant relative to suspension culture cells. Mixed populations of poly(A) RNA from these tissues and cells allowed the determination of shared sequences among them. When all combinations of poly(A) RNA were mixed, the percentage of ;single' copy DNA that saturated was equivalent to diverse sequence complexity estimates of about 60,000. When mixed poly(A) RNA from suspension cells from (+) and (-) auxin medium were compared, they shared about 40,000 sequences and (-) auxin cells contained an additional 20,000. Both (+) and (-) tissue culture cells shared a subset of about 20,000 sequences with cells from (+) and (-) auxin treated hypocotyl. A third subset of about 20,000 sequences was shared by (-) auxin suspension cells and hypocotyl treated with or without auxin, a subset most of which were not shared by (+) auxin suspension cells. Kinetic and saturation data estimates of poly(A) RNA complexity compared favorably and indicated that exogenous auxin treatment can dramatically alter the complexity of all classes of poly(A) RNAs in cultured cells.

Journal Article↗
PubMed2026

Thalamic NRXN1-mediated input to human cortical progenitors drives excitatory neurogenesis.

The human cerebral cortex develops through coordinated signals from within the cortex and from other brain regions, including the thalamus. However, how thalamic neuronal projections influence early human cortical development remains less well-understood. In this study, we fused cortical and thalamic organoids to investigate how thalamic input shapes the maturation of human cortical cells. Using single-nuclei RNA-sequencing and cellular imaging, we found that thalamic input increases the production of cortical excitatory neurons. We identify neurexin-1 (NRXN1) as a mediator of physical contact between thalamic axons and cortical outer radial glia. Genetic knockout of thalamic NRXN1 reduced these contacts and attenuated the production of upper-layer excitatory neurons. These findings reveal a mechanism by which thalamic input regulates human cortical progenitors and shapes excitatory neuron production during development.

Animals↗
PubMed1985

Characterization of the in vivo RNA product of the pOUT promoter of IS10R.

We characterized a single RNA species (RNAout1) which was the major in vivo RNA made from pOUT of IS10R. RNAout1 was 70 nucleotides long; its 5' end corresponded exactly to the in vitro start of pOUT transcription. The concentration of RNAout1 was estimated at 5 to 10 molecules per cell containing the single-copy plasmid NR1. RNA sequences from pOUT of IS10L were detected at a much lower (less than one molecule per cell) steady-state concentration and may be preferentially degraded in vivo. We suggest that the low level of the IS10L transcript led to the inability of IS10L sequences to translationally inhibit Tn10 transposition.

Base Sequence↗
PubMed2025

Single-Cell Splicing Isoform Atlas of the Adult Human Heart and Heart Failure.

BACKGROUND: Alternative splicing plays crucial roles in normal heart development and cardiac disease by influencing protein-coding sequences, functional domains, and molecular networks. However, a detailed characterization of the human heart isoform landscape remains incomplete. METHODS: Leveraging long-read single-nucleus RNA sequencing and computational analysis, we dissected full-length isoform heterogeneities, expression patterns, and usage shifts across cell types, cell states, and cardiac conditions of the adult left ventricle. We applied in silico approaches to assess the functional relevance of identified isoforms; validated isoform compositions of representative cardiac genes using reverse transcription quantitative polymerase chain reaction and targeted amplicon sequencing; and developed a web server for interactive navigation of our results. RESULTS: The data revealed that isoform heterogeneity is widespread in the cardiac cellular system, serving as a posttranscriptional buffer mechanism that calibrates the molecule reservoirs in human hearts. In healthy left ventricles, ≈30% of cell type-specific genes were polyform, using multiple isoforms tailored to cell type-specific programs. Among ubiquitously expressed genes, >300 showed differential isoform usage with cell type specificity in normal hearts. Comparisons of cardiomyocytes across conditions uncovered 379 genes with marked isoform usage shifts, most of which are predicted to change protein coding outcomes through direct changes in protein coding sequences and switches between intron retention and non-protein-coding biotypes. In contrast, cell state-specific programs tend to operate on monoform genes associated with changes among cell states. In addition, our data revealed heart failure-associated differential isoform usage events in stromal and immune cell types in the cardiac microenvironment. CONCLUSIONS: We present a comprehensive atlas of splicing isoforms in the normal adult heart and heart failure through long-read single-nucleus RNA sequencing and computational analyses. The results suggest crucial roles of isoforms in buffering core cellular programs and contributing to disease-associated cell states. The full-length details of these cell-specific isoforms serve as an important reference for downstream translational and mechanistic studies and are available on our online data portal at https://github.com/gaolabtools/heart-isoform-atlas.

Humans↗
PubMed1988

A rapid and sensitive solution hybridisation assay for the quantitative determination of specific viral RNA sequences.

A solution hybridisation assay has been developed which allows the quantitation of specific viral (RNA) sequences in infected cells. The assay makes use of single-stranded (ss) RNA probes of known polarity synthesised at high specific activity in vitro from cDNA clones of the relevant viral gene by the SP6 or T7 RNA polymerase. These probes are used together with samples containing the RNA to be detected at a known concentration to construct a calibration curve to relate RNase resistant radioactivity following solution hybridisation to amount of RNA. The amount of RNA in experimental samples is then determined using the calibration curve that is produced each time the assay is performed. The UKtc strain of Rotavirus growing in BSC-1 cells was used to develop this method but with the substitution of suitable cDNA clones it could be applied to any viral system.

DNA↗