Single-cell mRNA Research Articles

Keratin 19 (Krt19) is a novel marker gene for epicardial cells.

Epicardial cells regulate heart growth by secreting numerous growth factors and undergoing lineage specification into other cardiac lineages. However, the lack of specific marker genes for epicardial cells has hindered the understanding of this cell type in heart development. Through the analysis of a cardiac single cell mRNA sequencing dataset, we identified a novel epicardial gene named Keratin 19 (Krt19). Further analysis of the expression patterns of Krt19 and Wt1, a well-known epicardial gene, revealed their preferences in major cardiac cell types. Using lineage-tracing analysis, we analyzed Krt19-CreER labeled cells at multiple time windows and found that it labels epicardial cells at both embryonic and neonatal stages. Furthermore, we studied the function of epicardial cells using a diphtheria toxin A chain (DTA)-based cell ablation system. We discovered that Krt19-CreER labeled cells are essential for fetal heart development. Finally, we investigated the function of Krt19-CreER and Wt1-CreER labeled cells in neonatal mouse development. We observed that the Krt19-CreER; Rosa-DTA mice displayed a smaller size after tamoxifen treatment, suggesting the potential importance of Krt19-CreER labeled cells in neonatal mouse development. Additionally, we found that Wt1-CreER; Rosa-DTA mice died at early stages, likely due to defects in the kidney and spleen. In summary, we have identified Krt19 as a new epicardial cell marker gene and further explored the function of epicardial cells using the Krt19-CreER and Wt1-CreER-mediated DTA ablation system.

What can we learn when fitting a simple telegraph model to a complex gene expression model?

In experiments, the distributions of mRNA or protein numbers in single cells are often fitted to the random telegraph model which includes synthesis and decay of mRNA or protein, and switching of the gene between active and inactive states. While commonly used, this model does not describe how fluctuations are influenced by crucial biological mechanisms such as feedback regulation, non-exponential gene inactivation durations, and multiple gene activation pathways. Here we investigate the dynamical properties of four relatively complex gene expression models by fitting their steady-state mRNA or protein number distributions to the simple telegraph model. We show that despite the underlying complex biological mechanisms, the telegraph model with three effective parameters can accurately capture the steady-state gene product distributions, as well as the conditional distributions in the active gene state, of the complex models. Some effective parameters are reliable and can reflect realistic dynamic behaviors of the complex models, while others may deviate significantly from their real values in the complex models. The effective parameters can also be applied to characterize the capability for a complex model to exhibit multimodality. Using additional information such as single-cell data at multiple time points, we provide an effective method of distinguishing the complex models from the telegraph model. Furthermore, using measurements under varying experimental conditions, we show that fitting the mRNA or protein number distributions to the telegraph model may even reveal the underlying gene regulation mechanisms of the complex models. The effectiveness of these methods is confirmed by analysis of single-cell data for E. coli and mammalian cells. All these results are robust with respect to cooperative transcriptional regulation and extrinsic noise. In particular, we find that faster relaxation speed to the steady state results in more precise parameter inference under large extrinsic noise.

Overexpressing S100A9 ameliorates NK cell dysfunction in estrogen receptor-positive breast cancer

BackgroundEstrogen receptor (ER) positive human epidermal growth factor receptor 2 (HER2) negative breast cancer (ER+/HER2−BC) and triple-negative breast cancer (TNBC) are two distinct breast cancer molecular subtypes, especially in tumor immune microenvironment (TIME). The TIME of TNBC is considered to be more inflammatory than that of ER+/HER2−BC. Natural killer (NK) cells are innate lymphocytes that play an important role of tumor eradication in TME. However, studies focusing on the different cell states of NK cells in breast cancer subtypes are still inadequate.MethodsIn this study, single-cell mRNA sequencing (scRNA-seq) and bulk mRNA sequencing data from ER+/HER2−BC and TNBC were analyzed. Key regulator of NK cell suppression in ER+/HER2−BC, S100A9, was quantified by qPCR and ELISA in MCF-7, T47D, MDA-MB-468 and MDA-MB-231 cell lines. The prognosis predictability of S100A9 and NK activation markers was evaluated by Kaplan–Meier analyses using TCGA-BRAC data. The phenotype changes of NK cells in ER+/HER2−BC after overexpressing S100A9 in cancer cells were evaluated by the production levels of IFN-gamma, perforin and granzyme B and cytotoxicity assay.ResultsBy analyzing scRNA-seq data, we found that multiple genes involved in cellular stress response were upregulated in ER+/HER2−BC compared with TNBC. Moreover, TLR regulation pathway was significantly enriched using differentially expressed genes (DEGs) from comparing the transcriptome data of ER+/HER2−BC and TNBC cancer cells, and NK cell infiltration high/low groups. Among the DEGs, S100A9 was identified as a key regulator. Patients with higher expression levels of S100A9 and NK cell activation markers had better overall survival. Furthermore, we proved that overexpression of S100A9 in ER+/HER2-cells could improve cocultured NK cell function.ConclusionIn conclusion, the study we presented demonstrated that NK cells in ER+/HER2−BC were hypofunctional, and S100A9 was an important regulator of NK cell function in ER+BC. Our work contributes to elucidate the regulatory networks between cancer cells and NK cells and may provide theoretical basis for novel drug development.

Understanding the Mechanisms of the Gβ5/R7-RGS Complex in Pain Transmission through Single Cell mRNA Profiling

The dorsal root ganglion (DRG) and trigeminal ganglion (TG) contain cell bodies of the peripheral sensory neurons that transmit pain signals from different parts of body to the central nervous system. Peripheral neuropathy and trigeminal neuralgia are common painful conditions that can be caused by pathologies affecting these nerves. Current clinical treatment of these conditions often relies on anticonvulsant drugs with significant neurological side effects. Lack of effective treatments of these conditions highlights the need for better therapeutic approaches. GNB5 encodes the protein Gβ5, a divergent member of the G protein β family, that binds with members (RGS6, 7, 9 and 11) of the R7 subfamily of the regulators of G signaling (R7-RGS) to form obligate Gβ5/R7-RGS complexes. The Gβ5/R7-RGS complex proteins are expressed primarily in neurons including those in the DRG and TG. They function as GTPase-Activating Proteins (GAP) to dampen signaling downstream of Gi/o-coupled receptors. Studies in our lab demonstrated that Adv-Cre+/−; Gnb5fl/fl mice lacking Gnb5 in sensory neurons have significantly reduced mechanical, thermal and chemical nociception. However, RGS7-Cre+/−; Gnb5fl/fl mice lacking Gnb5 in Rgs7 expressing neurons exhibited only diminished mechanical nociception. Additionally, treatment with GABA-B receptor antagonist 2- hydroxysaclofen abolished the inhibition of mechanical nociception in both Advillin- Cre+/-; Gnb5fl/fl and Rgs7-Cre+/-; Gnb5fl/fl mice, implicating the involvement of GABA-B receptor signaling in Gβ5/Rgs7-associated mechanical nociception. To further understand the cellular and molecular mechanisms of Gβ5/R7-RGS complex-mediated nociception, we analyzed and compared the expression profiles of the Gβ5/R7-RGS genes in DRG and TG through bioinformatic analysis of publicly available scRNAseq datasets and in situ hybridization of selected genes using RNAscope technology. We found high levels of Gnb5 expression across all sensory neuron cell types while the expressions of members of R7-RGS subfamily showed distinctive patterns among these neuron cell types. Single cell clustering analysis using known neuronal markers revealed that Rgs7 and Rgs11 were highly expressed in majority of neuron types, while Rgs6 and Rgs9 were represented in smaller, more distinct clusters. These results suggest that particular members of R7-RGS gene family might play distinct roles in different types of nociception, a finding that warrants further investigation into the individual mechanisms of each member of this gene family in pain transmission. NIDDK Intramural Research Program ZIA DK043304-29. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Enhanced Annotation of CD45RA to Distinguish T cell Subsets in Single Cell RNA-seq via Machine Learning

Abstract T cell heterogeneity presents a challenge for accurate cell identification, understanding their inherent plasticity, and characterizing their critical role in adaptive immunity. With the advent of single-cell RNA sequencing (scRNA-seq), researchers can now investigate the gene expression profiles of these surface proteins at the single-cell level, having a deeper understanding of cell identity. However, CD45RA, the isoform of CD45 which distinguish between naive/central memory T cells and effector memory/effector memory cells re-expressing CD45RA T cells, cannot be well profiled by scRNA-seq due to the difficulty in mapping short reads to genes. In order to facilitate cell type annotation in T cell scRNA-seq analysis, we employed machine learning and trained a CD45RA+/- classifier on single-cell mRNA count data annotated with known CD45RA antibody levels provided by cellular indexing of transcriptomes and epitopes sequencing (CITE-seq) data. Among all algorithms we tested, the trained support vector machine (SVM) with a radial basis function (RBF) kernel with optimized hyperparameters achieved a 99.96% accuracy on an unseen dataset. The multilayer Perceptron (MLP) classifier, the second most predictive method overall, also achieved a decent accuracy of 99.74%. Our simple yet robust machine learning approach provides a valid inference on the CD45RA level, assisting the cell identity annotation and further exploring the heterogeneity within human T cells.

Deciphering the heterogeneity dominated by tumor-associated macrophages for survival prognostication and prediction of immunotherapy response in lung adenocarcinoma

Tumor-associated macrophages (TAMs) are a specific subset of macrophages that reside inside the tumor microenvironment. The dynamic interplay between TAMs and tumor cells plays a crucial role in the treatment response and prognosis of lung adenocarcinoma (LUAD). The study aimed to examine the association between TAMs and LUAD to advance the development of targeted strategies and immunotherapeutic approaches for treating this type of lung cancer. The study employed single-cell mRNA sequencing data to characterize the immune cell composition of LUAD and delineate distinct subpopulations of TAMs. The “BayesPrism” and “Seurat” R packages were employed to examine the association between these subgroups and immunotherapy and clinical features to identify novel immunotherapy biomarkers. Furthermore, a predictive signature was generated to forecast patient prognosis by examining the gene expression profile of immunotherapy-associated TAMs subsets and using 104 machine-learning techniques. A comprehensive investigation has shown the existence of a hitherto unidentified subgroup of TAMs known as RGS1 + TAMs, which has been found to have a strong correlation with the efficacy of immunotherapy and the occurrence of tumor metastasis in LUAD patients. CD83 was identified CD83 as a distinct biomarker for the expression of RGS1 + TAMs, showcasing its potential utility as an indicator for immunotherapeutic interventions. Furthermore, the prognostic capacity of the RTMscore signature, encompassing three specific mRNA (NR4A2, MMP14, and NPC2), demonstrated enhanced robustness when contrasted against the comprehensive collection of 104 features outlined in the published study. CD83 has potential as an immunotherapeutic biomarker. Meanwhile, The RTMscore signature established in the present study might be beneficial for survival prognostication.

Chronic Opioid Treatment Arrests Neurodevelopment and Alters Synaptic Activity in Human Midbrain Organoids.

Understanding the impact of long-term opioid exposure on the embryonic brain is critical due to the surging number of pregnant mothers with opioid dependency. However, this has been limited by human brain inaccessibility and cross-species differences in animal models. Here, a human midbrain model is established that uses hiPSC-derived midbrain organoids to assess cell-type-specific responses to acute and chronic fentanyl treatment and fentanyl withdrawal. Single-cell mRNA sequencing of 25,510 cells from organoids in different treatment groups reveals that chronic fentanyl treatment arrests neuronal subtype specification during early midbrain development and alters synaptic activity and neuron projection. In contrast, acute fentanyl treatment increases dopamine release but does not significantly alter gene expression related to cell lineage development. These results provide the first examination of the effects of opioid exposure on human midbrain development at the single-cell level.

Sexual dimorphism in melanocyte stem cell behavior reveals combinational therapeutic strategies for cutaneous repigmentation

Vitiligo is an autoimmune skin disease caused by cutaneous melanocyte loss. Although phototherapy and T cell suppression therapy have been widely used to induce epidermal re-pigmentation, full pigmentation recovery is rarely achieved due to our poor understanding of the cellular and molecular mechanisms governing this process. Here, we identify unique melanocyte stem cell (McSC) epidermal migration rates between male and female mice, which is due to sexually dimorphic cutaneous inflammatory responses generated by ultra-violet B exposure. Using genetically engineered mouse models, and unbiased bulk and single-cell mRNA sequencing approaches, we determine that manipulating the inflammatory response through cyclooxygenase and its downstream prostaglandin product regulates McSC proliferation and epidermal migration in response to UVB exposure. Furthermore, we demonstrate that a combinational therapy that manipulates both macrophages and T cells (or innate and adaptive immunity) significantly promotes epidermal melanocyte re-population. With these findings, we propose a novel therapeutic strategy for repigmentation in patients with depigmentation conditions such as vitiligo.

P148 Longitudinal single-cell data informs deterministic modelling of inflammatory bowel disease

Abstract Background Crucial contributions to our understanding of the inflammatory process in inflammatory bowel disease (IBD) have been made using single cell level approaches. Extracting mechanistic details of cell and gene dynamics would be critical to understanding the mechanisms mediating mucosal healing and predicting response to specific therapies. However, this remains challenging. Therefore, our aim was to use ordinary differential equations (ODEs) for mathematical modelling of cell abundance and prediction of biological processes in IBD. Methods We used mathematical modelling of published longitudinal single-cell mRNA sequencing (scRNA-seq) data from DSS colitis in mice to simulate cellular dynamics during IBD. We validated our predictions using pseudo-bulk gene expression analysis and flow cytometry. We also analysed a published microarray dataset from colonic biopsies of 51 IBD patients prior to infliximab treatment. Results Based on the scRNA-seq data, we used our mathematical model to predict the immune dynamics of a different mouse colitis protocol. We validated these predictions experimentally using flow cytometry and found good agreement between model simulation and experimental data (predicted vs. observed, Pearson's rho=0.92). We found that the estimated turnover rates predicted the protein level of the proliferation marker KI-67 (rho=0.88) in DSS colitis. Finally, we wanted to investigate whether our mathematical model reflected processes in human IBD. We reasoned that genes correlated with epithelial cell dynamics might be indicative of mucosal healing after DSS retrieval. We found more highly correlated genes when using simulated epithelial abundance compared to experimentally measured epithelial abundance. Many genes associated with tight junctions (Cldn8, Cftr, Crb3, Jam2) were among the highly positively correlated genes and genes associated with inflammation (Il1b, Jak2, Il10ra) were among the negatively correlated genes with epithelial cell abundance. We then looked for differentially expressed (DE) genes in pre-treatment colon biopsies between responders and non-responders to infliximab. We found a significant overlap of 25 positively correlated genes with upregulated DE genes in responders, i.e. associated with tight junctions, and 73 negatively correlated genes with upregulated genes in non-responders, i.e. associated with inflammatory pathways. Conclusion We demonstrate that mathematical modelling of colonic cell dynamics provides insight into IBD progression and treatment response. Furthermore, our results suggest that pre-treatment barrier integrity may predict and influence response to infliximab treatment.

Morphological, molecular, and functional characterization of mouse glutamatergic myenteric neurons.

The enteric nervous system (ENS) functions largely independently of the central nervous system (CNS). Glutamate, the dominant neurotransmitter in the CNS and sensory afferents, is not a primary neurotransmitter in the ENS. Only a fraction (∼2%) of myenteric neurons in the mouse distal colon and rectum (colorectum) are positive for vesicular glutamate transporter type 2 (VGLUT2), the structure and function of which remain undetermined. Here, we systematically characterized VGLUT2-positive enteric neurons (VGLUT2-ENs) through sparse labeling with adeno-associated virus, single-cell mRNA sequencing (scRNA-seq), and GCaMP6f calcium imaging. Our results reveal that the majority of VGLUT2-ENs (29 of 31, 93.5%) exhibited Dogiel type I morphology with a single aborally projecting axon; most axons (26 of 29, 89.7%) are between 4 and 10 mm long, each traversing 19 to 34 myenteric ganglia. These anatomical features exclude the VGLUT2-ENs from being intrinsic primary afferent or motor neurons. The scRNA-seq conducted on 52 VGLUT2-ENs suggests different expression profiles from conventional descending interneurons. Ex vivo GCaMP6f recordings from flattened colorectum indicate that almost all VGLUT2-EN (181 of 215, 84.2%) are indirectly activated by colorectal stretch via nicotinic cholinergic neural transmission. In conclusion, VGLUT2-ENs are a functionally unique group of enteric neurons with single aborally projecting long axons that traverse multiple myenteric ganglia and are activated indirectly by colorectal mechanical stretch. This knowledge will provide a solid foundation for subsequent studies on the potential interactions of VGLUT2-EN with extrinsic colorectal afferents via glutamatergic neurotransmission.NEW & NOTEWORTHY We reveal that VGLUT2-positive enteric neurons (EN), although constituting a small fraction of total EN, are homogeneously expressed in the myenteric ganglia, with a slight concentration at the intermediate region between the colon and rectum. Through anatomic, molecular, and functional analyses, we demonstrated that VGLUT2-ENs are activated indirectly by noxious circumferential colorectal stretch via nicotinic cholinergic transmission, suggesting their participation in mechanical visceral nociception.

Single-Cell RNA Sequencing Reveals Alterations in Patient Immune Cells with Pulmonary Long COVID-19 Complications.

Since the emergence of the COVID-19 pandemic, the effects of SARS-CoV-2 have been extensively researched. While much is already known about the acute phase of the infection, increasing attention has turned to the prolonged symptoms experienced by a subset of individuals, commonly referred to as long COVID-19 patients. This study aims to delve deeper into the immune landscape of patients with prolonged symptoms by implementing single-cell mRNA analysis. A 71-year-old COVID-19 patient presenting with persistent viral pneumonia was recruited, and peripheral blood samples were taken at 3 and 2 years post-acute infection onset. Patients and control peripheral blood mononuclear cells (PBMCs) were isolated and single-cell sequenced. Immune cell population identification was carried out using the ScType script. Three months post-COVID-19 patients' PBMCs contained a significantly larger immature neutrophil population compared to 2-year and control samples. However, the neutrophil balance shifted towards a more mature profile after 18 months. In addition, a notable increase in the CD8+ NKT-like cells could be observed in the 3-month patient sample as compared to the later one and control. The subsequent change in these cell populations over time may be an indicator of an ongoing failure to clear the SARS-CoV-2 infection and, thus, lead to chronic COVID-19 complications.

Single-Cell Imaging of mRNA by Target RNA-Initiated RCA.

We present a powerful method for direct mRNA detection based on ligation-based recognition and in situ amplification, capable of single-cell imaging mRNA at single-nucleotide and single-molecule resolution. Attributed to the use of Splint R ligase that can ligate padlock probe with RNA as target template, this method can efficiently detect mRNA in the absence of reverse transcription. This method enables spatial localization and correlation analysis of gene expression in single cells, which helps us to elucidate gene function and regulatory mechanisms.

In situ Patch-seq analysis of microglia reveals a lack of stress genes as found in FACS-isolated microglia.

We applied the patch-seq technique to harvest transcripts from individual microglial cells from cortex, hippocampus and corpus callosum of acute brain slices from adult mice. After recording membrane currents with the patch-clamp technique, the cytoplasm was collected via the pipette and underwent adapted SMART-seq2 preparation with subsequent sequencing. On average, 4138 genes were detected in 113 cells from hippocampus, corpus callosum and cortex, including microglia markers such as Tmem119, P2ry12 and Siglec-H. Comparing our dataset to previously published single cell mRNA sequencing data from FACS-isolated microglia indicated that two clusters of cells were absent in our patch-seq dataset. Pathway analysis of marker genes in FACS-specific clusters revealed association with microglial activation and stress response. This indicates that under normal conditions microglia in situ lack transcripts associated with a stress-response, and that the microglia-isolation procedure by mechanical dissociation and FACS triggers the expression of genes related to activation and stress.

Cardiomyocyte-derived USP28 negatively regulates antioxidant response and promotes cardiac hypertrophy via deubiquitinating TRIM21.

Rationale: Cardiac hypertrophy is an important pathological basis for heart failure. Most physiological activities of cardiomyocytes are regulated by proteins and their post-translational modification. Deubiquitinating enzymes (DUBs) are involved in protein stability maintenance and closely related to myocardial hypertrophy. In this study, we aimed to clarify the regulatory role of a DUB, ubiquitin-specific peptidase 28 (USP28), in cardiac hypertrophy and explore the molecular mechanism behind. Methods: Transcriptome and single-cell mRNA sequencing was used to demonstrate the association of USP28 and cardiac hypertrophy. Cardiomyocyte-specific USP28 knockout mice (USP28CKO) were subjected to angiotensin II (Ang II) infusion or transverse aortic constriction (TAC) models. Coimmunoprecipitation combined mass spectrum analysis (Co-IP/MS) was applied to screen out the substrate of USP28. Results: We first showed the up-regulation of USP28 in cardiac hypertrophy, and its cellular localization of cardiomyocytes. USP28CKO protects mouse heart against Ang II- or TAC-induced cardiac dysfunction and hypertrophy. Mechanistically, we identified tripartite motif-containing protein 21 (TRIM21) as the potential substrate of USP28 by Co-IP/MS analysis. Cardiomyocyte USP28 deubiquitinates and stabilizes TRIM21 to negatively regulate nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response, increasing oxidative stress in cardiomyocytes and promoting cardiac hypertrophy and injury. Finally, using a selective USP28 inhibitor Otilonium Bromide, we confirmed the therapeutic effect of pharmacological inhibition of USP28 against TAC-induced established hypertrophic heart failure. Conclusion: Our study illustrates a cardiomyocyte-specific USP28-TRIM21 axis in regulating hypertrophic cardiomyopathy and presents USP28 as a potential target for the treatment of cardiac hypertrophy.

Specific cell subclusters of dental pulp stem cells respond to distinct pathogens through the ROS pathway.

Microbial pathogens invade various human organs, including the oral cavity. Candida albicans (C.a) and Streptococcus mutans (S.m) served respectively as representative oral pathogenic fungi and bacteria to stimulate dental pulp stem cells (DPSCs) and to screen the DPSC subcluster that specifically responded to fungal infection. DPSCs were obtained from the impacted third molars of six healthy subjects. Then, cells were mixed and divided into three samples, two of which were stimulated with C.a and S.m, respectively; the third sample was exposed to cell medium only (Ctrl). Single-cell mRNA sequencing analysis of treated DPSCs was performed. DPSCs were composed of four major clusters of which one, DPSC.7, exhibited unique changes compared to those of other subclusters. The DPSC.7 cell percentage of the C.a sample was twice those of the Ctrl and S.m samples. DPSC.7 cells expressed genes associated with the response to reactive oxygen species (ROS) response. DPSC.7 subgroup cells established characteristic aggregation under the stimulation of different pathogens in UMAP. The MAPK/ERK1/2 and NF-κB pathways were up-regulated, DUSP1/5/6 expressions were suppressed, FOS synthesis was activated, the immune-related pathway was induced, and the levels of cytokines, including IL-6 and CCL2, were up-regulated in DPSC.7 cells when stimulated with C.a. Our study analyzed the cellular and molecular properties of DPSCs infected by oral fungi and bacteria with single-cell RNA sequencing. A subcluster of DPSCs responded specifically to infections with different pathogens, activating the MAPK and NF-κB pathways to induce immune responses via the ROS pathway. This suggests novel treatment strategies for fungal infections.

Systems biology analysis reveals distinct molecular signatures associated with immune responsiveness to the BNT162b COVID-19 vaccine

Human immune responses to COVID-19 vaccines display a large heterogeneity of induced immunity and the underlying immune mechanisms for this remain largely unknown. Using a systems biology approach, we longitudinally profiled a unique cohort of female high and low responders to the BNT162b vaccine, who were known from previous COVID-19 vaccinations to develop maximum and minimum immune responses to the vaccine. We utilized high dimensional flow cytometry, bulk and single cell mRNA sequencing and 48-plex serum cytokine analyses. We revealed early, transient immunological and molecular signatures that distinguished high from low responders and correlated with B and T cell responses measured 14 days later. High responders featured a distinct transcriptional activity of interferon-driven genes and genes connected to enhanced antigen presentation. This was accompanied by a robust cytokine response related to Th1 differentiation. Both transcriptome and serum cytokine signatures were confirmed in two independent confirmatory cohorts. Collectively, our data contribute to a better understanding of the immunogenicity of mRNA-based COVID-19 vaccines, which might lead to the optimization of vaccine designs for individuals with poor vaccine responses. German Center for Infection Research, German Center for Lung Research, German Research Foundation, Excellence Strategy EXC 2155 "RESIST" and European Regional Development Fund.

Single-cell profiling of bronchoalveolar cells reveals a Th17 signature in neutrophilic severe equine asthma.

Severe equine asthma (SEA) is a complex respiratory condition characterized by chronic airway inflammation. It shares many clinical and pathological features with human neutrophilic asthma, making it a valuable model for studying this condition. However, the immune mechanisms driving SEA have remained elusive. Although SEA has been primarily associated with a Th2 response, there have also been reports of Th1, Th17, or mixed-mediated responses. To uncover the elusive immune mechanisms driving SEA, we performed single-cell mRNA sequencing (scRNA-seq) on cryopreserved bronchoalveolar cells from 11 Warmblood horses, 5 controls and 6 with SEA. We identified six major cell types, including B cells, T cells, monocytes-macrophages, dendritic cells, neutrophils, and mast cells. All cell types exhibited significant heterogeneity, with previously identified and novel cell subtypes. Notably, we observed monocyte-lymphocyte complexes and detected a robust Th17 signature in SEA, with CXCL13 upregulation in intermediate monocytes. Asthmatic horses exhibited expansion of the B-cell population, Th17 polarization of the T-cell populations, and dysregulation of genes associated with T-cell function. Neutrophils demonstrated enhanced migratory capacity and heightened aptitude for neutrophil extracellular trap formation. These findings provide compelling evidence for a predominant Th17 immune response in neutrophilic SEA, driven by dysregulation of monocyte and T-cell genes. The dysregulated genes identified through scRNA-seq have potential as biomarkers and therapeutic targets for SEA and provide insights into human neutrophilic asthma.

Exact solution of a three-stage model of stochastic gene expression including cell-cycle dynamics

The classical three-stage model of stochastic gene expression predicts the statistics of single cell mRNA and protein number fluctuations as a function of the rates of promoter switching, transcription, translation, degradation and dilution. While this model is easily simulated, its analytical solution remains an unsolved problem. Here we modify this model to explicitly include cell-cycle dynamics and then derive an exact solution for the time-dependent joint distribution of mRNA and protein numbers. We show large differences between this model and the classical model which captures cell-cycle effects implicitly via effective first-order dilution reactions. In particular we find that the Fano factor of protein numbers calculated from a population snapshot measurement are underestimated by the classical model whereas the correlation between mRNA and protein can be either over- or underestimated, depending on the timescales of mRNA degradation and promoter switching relative to the mean cell-cycle duration time.

Analysis of cell-specific transcriptional responses in human colon tissue using CIBERSORTx

Diet is an important determinant of overall health, and has been linked to the risk of various cancers. To understand the mechanisms involved, transcriptomic responses from human intervention studies are very informative. However, gene expression analysis of human biopsy material only represents the average profile of a mixture of cell types that can mask more subtle, but relevant cell-specific changes. Here, we use the CIBERSORTx algorithm to generate single-cell gene expression from human multicellular colon tissue. We applied the CIBERSORTx to microarray data from the PHYTOME study, which investigated the effects of different types of meat on transcriptional and biomarker changes relevant to colorectal cancer (CRC) risk. First, we used single-cell mRNA sequencing data from healthy colon tissue to generate a novel signature matrix in CIBERSORTx, then we determined the proportions and gene expression of each separate cell type. After comparison, cell proportion analysis showed a continuous upward trend in the abundance of goblet cells and stem cells, and a continuous downward trend in transit amplifying cells after the addition of phytochemicals in red meat products. The dietary intervention influenced the expression of genes involved in the growth and division of stem cells, the metabolism and detoxification of enterocytes, the translation and glycosylation of goblet cells, and the inflammatory response of innate lymphoid cells. These results show that our approach offers novel insights into the heterogeneous gene expression responses of different cell types in colon tissue during a dietary intervention.

Following the Birth, Life, and Death of mRNAs in Single Cells.

Recent advances in single-molecule imaging of mRNAs in fixed and living cells have enabled the lives of mRNAs to be studied with unprecedented spatial and temporal detail. These approaches have moved beyond simply being able to observe specific events and have begun to allow an understanding of how regulation is coupled between steps in the mRNA life cycle. Additionally, these methodologies are now being applied in multicellular systems and animals to provide more nuanced insights into the physiological regulation of RNA metabolism.