Major Cell Types Research Articles

Single-cell Transcriptomics Reveals Activation of Macrophages in All-trans Retinoic Acid (atRA)-induced Cleft Palate.

Cleft palate is among the most common birth defects with an impact on swallowing and speaking and is difficult to diagnose with ultrasound during pregnancy. In this study, we systematically capture the cellular composition of all-trans retinoic acid (atRA)-exposed and normal embryonic gestation 16.5 days mouse palate by the single-cell RNA sequencing technique. The authors identified 14 major cell types with the largest proportion of fibroblasts. The proportion of myeloid cells in atRA-exposed palate was markedly higher than those in the normal palate tissue, especially M1-like macrophages and monocytes. The upregulated genes of the different expression genes between atRA-exposed palate and normal palate tissue were linked to the biological processes of leukocyte chemotaxis and migration. Protein TLR2, CXCR4, THBS1, MRC1, transcription factor encoding genes Cebpb, Fos, Jun, Rela, and signaling pathway IL-17 and phagosome were found to be significantly involved in these processes. Subsequently, cellular communication network analysis suggested that myeloid-centered cell interactions SELL, SELPLG, MIF, CXCL, ANNEXIN, THBS, and NECTIN were significantly more activated in atRA-exposed palate. Overall, we delineate the single-cell landscape of atRA-induced cleft palate, revealing the effects of overexposure to atRA during palate tissue development and providing insights for the diagnosis of cleft palate.

Defining the molecular fingerprint of bladder and kidney fibroblasts.

Fibroblasts are integral to the organization and function of all organs and play critical roles in pathologies such as fibrosis; however, we have limited understanding of the fibroblasts that populate the bladder and kidney. In this review, I describe how transcriptomics is leading to a revolution in our understanding of fibroblast biology by defining the molecular fingerprint (i.e., transcriptome) of universal and specialized fibroblast types, revealing gene signatures that allows one to resolve fibroblasts from other mesenchymal cell types, and providing a new comprehension of the fibroblast lineage. In the kidney, transcriptomics is giving us new insights into the molecular fingerprint of kidney fibroblasts, including those for cortical fibroblasts, medullary fibroblasts, and erythropoietin (EPO)-producing Norn fibroblasts, as well as new information about the gene signatures of kidney myofibroblasts and the transition of kidney fibroblasts into myofibroblasts. Transcriptomics has also revealed that the major cell type in the bladder interstitium is the fibroblast, and that multiple fibroblast types, each with their own molecular fingerprint, are found in the bladder wall. Interleaved throughout is a discussion of how transcriptomics can drive our future understanding of fibroblast identification, diversity, function, and their roles in bladder and kidney biology and physiology in health and in disease states.

Single-cell transcriptome analysis of the germ cells and somatic cells during mitotic quiescence stage in goats.

The mitotic quiescence of prospermatogonia is the event known to occur during genesis of the male germline and is tied to the development of the spermatogenic lineage. The regulatory mechanisms and the functional importance of this process have been demonstrated in mice; however, regulation of this process in human and domestic animal is still largely unknown. In this study, we employed single-cell RNA sequencing to identify transcriptional signatures of prospermatogonia and major somatic cell types in testes of goats at E85, E105, and E125. We identified both common and specific Gene Ontology categories, transcription factor regulatory networks, and cell-cell interactions in cell types from goat testis. We also analyzed the transcriptional dynamic changes in prospermatogonia, Sertoli cells, Leydig cells, and interstitial cells. Our datasets provide a useful resource for the study of domestic animal germline development.

Regulation of extracellular progranulin in medial prefrontal cortex

Progranulin is a secreted pro-protein that has anti-inflammatory and neurotrophic effects and is necessary for maintaining lysosomal function. Mutations in progranulin (GRN) are a major cause of frontotemporal dementia. Most pathogenic GRN mutations cause progranulin haploinsufficiency, so boosting progranulin levels is a promising therapeutic strategy. Progranulin is constitutively secreted, then taken up and trafficked to lysosomes. Before being taken up from the extracellular space, progranulin interacts with receptors that may mediate anti-inflammatory and growth factor-like effects. Modifying progranulin trafficking is a viable approach to boosting progranulin, but progranulin secretion and uptake by cells in the brain is poorly understood and may involve distinct mechanisms from other parts of the body. Understanding the cell types and processes that regulate extracellular progranulin in the brain could provide insight into progranulin's mechanism of action and inform design of progranulin-boosting therapies. To address this question we used microdialysis to measure progranulin in interstitial fluid (ISF) of mouse medial prefrontal cortex (mPFC). Grn+/− mice had approximately 50% lower ISF progranulin than wild-type mice, matching the reduction of progranulin in cortical tissue. Fluorescent in situ hybridization and immunofluorescence confirmed that microglia and neurons are the major progranulin-expressing cell types in the mPFC. Studies of conditional microglial (Mg-KO) and neuronal (N-KO) Grn knockout mice revealed that loss of progranulin from either cell type results in approximately 50% reduction in ISF progranulin. LPS injection (i.p.) produced an acute increase in ISF progranulin in mPFC. Depolarizing cells with KCl increased ISF progranulin, but this response was not altered in N-KO mice, indicating progranulin secretion by non-neuronal cells. Increasing neuronal activity with picrotoxin did not increase ISF progranulin. These data indicate that microglia and neurons are the source of most ISF progranulin in mPFC, with microglia likely secreting more progranulin per cell than neurons. The acute increase in ISF progranulin after LPS treatment is consistent with a role for extracellular progranulin in regulating inflammation, and may have been driven by microglia or peripheral immune cells. Finally, these data indicate that mPFC neurons engage in constitutive progranulin secretion that is not acutely changed by neuronal activity.

Machine learning reveals PANoptosis as a potential reporter and prognostic revealer of tumour microenvironment in lung adenocarcinoma.

Lung adenocarcinoma (LUAD), a prominent lung cancer subtype, has an underexplored relationship with PANoptosis, a recently discovered mode of tumour cell death. This study incorporated iron death, copper death, scorch death, necrotizing apoptosis and bisulfide death into a pan-death gene set (PANoptosis) and conducted single-cell analysis of scRNA-seq data from 11 LUAD samples. Differentially expressed genes were identified, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed. Univariate COX regression and least absolute shrinkage and selection operator (LASSO) regression were used to screen PANoptosis key genes for constructing an LUAD risk model. The model's prognostic performance was evaluated using survival curves, risk scores and validation in the Gene Expression Omnibus database. The study also explored the correlation between risk scores, tumour biological function, immunotherapy, drug sensitivity and immune infiltration. The SMS gene in the PANoptosis model was silenced in two LUAD cell lines for cellular validation. Single-cell analysis revealed eight major cell types and several PANoptosis genes significantly associated with LUAD survival. The risk model demonstrated strong prognostic performance and association with immune infiltration, suggesting PANoptosis involvement in LUAD tumour immunity. Cellular validation further supported these findings. The PANoptosis key risk genes are believed to be closely related to the tumour microenvironment and immune regulation of LUAD, potentially providing valuable insights for early diagnosis and clinical treatment, and broader applications in other tumours and complex diseases.

Single Cell Transcriptomics Identifies Distinct Choroid Cell Populations Involved in Visually Guided Eye Growth.

Postnatal ocular growth is regulated by a vision-dependent mechanism, termed emmetropization, which acts to minimize refractive error through coordinated growth of the ocular tissues. Many studies suggest that the ocular choroid participates in the emmetropization process via the production of scleral growth regulators that control ocular elongation and refractive development. To elucidate the role of the choroid in emmetropization, we used single-cell RNA sequencing (scRNA-seq) to characterize the cell populations in the chick choroid and compare gene expression changes in these cell populations during conditions in which the eye is undergoing emmetropization. UMAP clustering analysis identified 24 distinct cell clusters in all chick choroids. 7 clusters were identified as fibroblast subpopulations; 5 clusters represented different populations of endothelial cells; 4 clusters were CD45+ macrophages, T cells and B cells; 3 clusters were Schwann cell subpopulations; and 2 clusters were identified as melanocytes. Additionally, single populations of RBCs, plasma cells and neuronal cells were identified. Significant changes in gene expression between control and treated choroids were identified in 17 cell clusters, representing 95% of total choroidal cells. The majority of significant gene expression changes were relatively small (< 2 fold). The highest changes in gene expression were identified in a rare cell population (0.11% - 0.49% of total choroidal cells). This cell population expressed high levels of neuron-specific genes as well as several opsin genes suggestive of a rare neuronal cell population that is potentially light sensitive. Our results, for the first time, provide a comprehensive profile of the major choroidal cell types and their gene expression changes during the process of emmetropization as well as insights into the canonical pathways and upstream regulators that coordinate postnatal ocular growth.

Deconvolution reveals cell-type-specific transcriptomic changes in the aging mouse brain

Mounting evidence highlights the crucial role of aging in the pathogenesis of Alzheimer's disease (AD). We have previously explored human apoE-targeted replacement mice across different ages and identified distinct molecular pathways driven by aging. However, the specific contribution of different brain cell types to the gene modules underlying these pathways remained elusive. To bridge this knowledge gap, we employed a computational deconvolution approach to examine cell-type-specific gene expression profiles in major brain cell types, including astrocytes (AS), microglia (MG), oligodendroglia (OG), neurons (NEU), and vascular cells (VC). Our findings revealed that immune module genes were predominantly expressed in MG, OG, and VC. The lipid metabolism module genes were primarily expressed in AS, MG, and OG. The mitochondria module genes showed prominent expression in VC, and the synapse module genes were primarily expressed in NEU and VC. Furthermore, we identified intra- and inter-cell-type interactions among these module genes and validated their aging-associated expression changes using published single cell studies. Our study dissected bulk brain transcriptomics data at the cellular level, providing a closer examination of the cell-type contributions to the molecular pathways driven by aging.

THU199 Using Primary Human Ovarian Stromal Cells To Elucidate Stromal Cell Influences On Egg Quality Across Pubertal Development

Abstract Disclosure: E. Tsui: None. M.M. Laronda: None. Follicles, the functional unit of the ovary with the potential to develop into mature eggs, are composed of a single germ cell and somatic support cells (i.e., granulosa and theca cells). While animal studies have demonstrated a positive effect of ovarian stromal cells to follicle development, the ovarian stromal compartment remains largely undescribed. Moreover, egg quality is maximized after the pubertal transition, but the impact of puberty on ovarian stromal cells and their influence on egg quality is not well understood. To elucidate the relationship between puberty, ovarian stromal cells, and follicle development, we utilized a follicle growth assay in which murine ovarian secondary follicles were cultured in three groups: alone (control), with pre-pubertal derived primary human ovarian stromal cells, or with post-pubertal derived primary human ovarian stromal cells. Follicles (n = 40 per group, N = 4 independent experiments) were monitored over 8 days by brightfield microscopy and evaluated for diameter and survival. No difference in follicle diameter or survival was detected between follicles grown with either pre-pubertal or post-pubertal derived stromal cells (diameter: p = 0.93 for days 2-8; survival: p &amp;gt; 0.9999 at day 8), however, each group of follicles co-cultured with stromal cells grew to larger diameters than follicles grown alone at days 2 (pre-pubertal and post-pubertal, p = 0.005), 4 (pre-pubertal, p = 0.0006; post-pubertal, p = 0.0002), 6 (pre-pubertal, p = 0.03; post-pubertal, p = 0.02), and 8 (pre-pubertal, p = 0.001; post-pubertal, p = 0.004). Survival between follicles grown alone or co-cultured with stromal cells trended towards significance (p = 0.0526 for both pre-pubertal and post-pubertal stroma groups), and all groups demonstrated average survival at day 8 above 70%. Follicle rupture was more commonly observed in follicles cultured with stromal cells (control, 10%; pre-pubertal, 40%; post-pubertal, 80%), with additional maturation studies in progress. Concurrently, the cellular landscape of ovarian stromal cells across puberty was evaluated with single cell RNA sequencing (n = 11, age range = 0.34 to 16.09 years, Tanner stages 1 and 3-5). Analyses revealed 12 sub-clusters representing five major cell types: endothelial cells, theca cells, vascular smooth muscle cells, granulosa cells, and immune cells. This data is screened through existing human secretome databases to identify candidate secreted factors that will be investigated in future follicle growth assays. These results demonstrate a benefit of human ovarian stromal cells in our follicle growth assay and, in conjunction with ongoing maturation and sequencing experiments, validate the utility of this assay to further understand how puberty affects the stromal microenvironment to impact egg quality, insight that can be used to develop and improve future reproductive technologies. Presentation: Thursday, June 15, 2023

FRI393 Mapping Endometrial Cell-type-specific Disease Signatures A nd Endometrial Organoids In Polycystic Ovary Syndrome

Abstract Disclosure: G. Eriksson: None. C. Li: None. S. Risal: None. H. Pui: None. S. Torstensson: None. A. Linden Hirschberg: None. S. Petropoulos: None. Q. Deng: None. E. Stener-Victorin: None. Women with polycystic ovary syndrome (PCOS) suffer from reduced fertility linked to implantation failure and miscarriage, as well as endometrial cancer, all associated with endometrial dysfunction. We hypothesize that cell-type-specific endometrial dysfunction in insulin-resistant and hyperandrogenic women with PCOS contributes to the observed endometrial dysfunction and that treatment aimed at improving insulin sensitivity and decreasing androgen excess has the potential to reverse identified alterations.To uncover cell-type-specific disease signatures and molecular pathways for PCOS-specific endometrial dysfunction, we first extracted single-nuclei (sn) from frozen endometrial biopsies taken in the proliferative phase (day 7-10) from controls (n=5) and women with PCOS (n=10) at baseline and in PCOS after 16 weeks of metformin (n=7) or lifestyle management (n=3) for snRNA-sequencing. The 10x Genomics protocol allowed us to sequence ≈10,000 nuclei/sample and ≈20,000 reads/nuclei. A total of 248,694 nuclei were captured and 6 major cell types were identified. The 3 largest cell clusters were i) stromal cells (124,055 nuclei), ii) epithelial cells (105,095 nuclei) and iii) immune cells (13,596), whilst iv) uterine smooth muscle cells, v) endothelial and vi) lymphoid cell clusters consisted of &amp;lt;3000 nuclei. Subsetting the epithelial cells revealed the functional luminal, glandular and ciliated cell types as well as proliferative cells. In the immune cell cluster, both myeloid and lymphoid lineage cells were identified, of which uterine NK-cells (uNK) and macrophages (uM) were the largest populations. Several differentially expressed genes (DEGs) of the epithelial subtypes, uNKs, uMs and stromal cells were identified in women with PCOS compared with controls. DEGs in cell types between PCOS cases and controls were enriched in pathways related to cilium organization in the ciliated epithelium, extracellular matrix structure in stromal cells, and cysteine-type endopeptidase activity in uM type 1. Of note, both 16-week of metformin treatment or lifestyle management restored multiple DEGs in each subtype. Second, to validate the identified molecular target, we have established 3D endometrial organoids (EOs) from fresh endometrial biopsies of control and PCOS women. After several passages, EOs were cryopreserved to create a biobank for future functional analyses of the molecular targets. The EOs can be re-established after thawing and immunofluorescence staining shows that the EOs consist of an intact proliferative basolateral epithelial membrane. This rigorous mapping of the human PCOS endometrium improves our understanding of cellular complexity and specific cell types. It provides new mechanistic insights into disease-specific endometrial dysfunction(s), and several of the identified dysfunctions can be reversed by current first-line interventions. Presentation: Friday, June 16, 2023

FRI443 Optimizing Conditions Of Tubule-like Structures In Testicular Organoids That Will Support Spermatogenesis

Abstract Disclosure: O. Printy: None. Testicular organoids are increasingly being investigated as a potential means of fertility restoration. These organoids have been shown to self-organize to recapitulate the inner architecture of the native testis, forming tubule-like structures that resemble the seminiferous tubules. However, these tubule-like structures lack dimensionality and do not contain a large, meiotically active germ cell population. We hypothesized that if testicular organoids are cultured under appropriate microenvironmental conditions, we will observe the development of tubule-like structures with diameters that resemble what is seen in vivo, in addition to appropriate somatic cell localization and Sertoli-germ cell interactions. To test this hypothesis, we generated organoids from 5dpp male CD1 mice at three cellular seeding densities: low density (170,000 cells per well), medium density (280,000 cells per well), and high density (310,000 cells per well). The heterogenous testicular cell suspension formed compact organoids at all three seeding densities and organoid diameter increased with increasing cell number. To assess the ability of the organoids to mimic the inner architecture of a testis, we performed immunohistochemistry on 14-day-old organoids: for the major somatic cell types of the testis Leydig cells (HSD3B2), peritubular myoid cells (ACTA1), and Sertoli cells (SOX9). Thus far, only medium density organoids appear to recapitulate the compartmentalization seen in native testes. These organoids tend to contain semi-continuous ring-like structures composed exclusively of ACTA1positive cells; these structures encapsulate a population of SOX9-positive cells, while HSD3B2-expressing cells are located on the periphery of the organoids. To assess the organoids’ ability to fuse to form a larger aggregate, organoids were transferred to 3D printed troughs. However, organoids did not merge and appeared to die. However, when organoids are cultured together in microcentrifuge tubes and alginate structures, they fused to form a larger aggregate. In the future, immunohistochemistry will be used to evaluate whether any extended tubule-like structures formed within the aggregates. Additionally, spermatogonial stem cells will be injected into the aggregates and ELISA assays will be performed to assess the spermatogenic and hormonal functionality of the organoids. Presentation: Friday, June 16, 2023

Single-Cell Transcriptomics Reveals Cellular Heterogeneity and Complex Cell-Cell Communication Networks in the Mouse Cornea.

To generate a single-cell RNA-sequencing (scRNA-seq) map and construct cell-cell communication networks of mouse corneas. C57BL/6 mouse corneas were dissociated to single cells and subjected to scRNA-seq. Cell populations were clustered and annotated for bioinformatic analysis using the R package "Seurat." Differential expression patterns were validated and spatially mapped with whole-mount immunofluorescence staining. Global intercellular signaling networks were constructed using CellChat. Unbiased clustering of scRNA-seq transcriptomes of 14,732 cells from 40 corneas revealed 17 cell clusters of six major cell types: nine epithelial cell, three keratocyte, two corneal endothelial cell, and one each of immune cell, vascular endothelial cell, and fibroblast clusters. The nine epithelial cell subtypes included quiescent limbal stem cells, transit-amplifying cells, and differentiated cells from corneas and two minor conjunctival epithelial clusters. CellChat analysis provided an atlas of the complex intercellular signaling communications among all cell types. We constructed a complete single-cell transcriptomic map and the complex signaling cross-talk among all cell types of the cornea, which can be used as a foundation atlas for further research on the cornea. This study also deepens the understanding of the cellular heterogeneity and heterotypic cell-cell interaction within corneas.

RNA-Seq-Based Molecular Classification Analyses in Colorectal Cancer and Synchronous Adenoma.

Colorectal cancers (CRC) are classified into consensus molecular subtypes (CMS) based on gene expression profiles. The revised classification system iCMS was proposed by considering intrinsic epithelial status, microsatellite instability (MSI), and fibrosis. This study aimed to provide molecular evidence for the adenoma-carcinoma sequence concept by examining CRC and synchronous adenomas using iCMS. Epithelial CMS cell proportion was estimated using CiberSortx, an in silico cell fractionation method that included CMS cell types among the reference cell types. A random forest (RF) model estimated the posterior probabilities of CMS classes, which were compared with the CiberSortx results. Gene expression profiles of the published iCMS signature panel were retrieved from our dataset and subjected to heatmap clustering for classification. Bulk RNA sequencing data were collected from 29 adenocarcinomas and 11 adenoma samples. CiberSortx showed all CRC contained either CMS2 or CMS3 as the major epithelial cancer cell type. The RF model classified approximately half of the CRC as CMS4, whereas CMS4 was hardly detected by CiberSortx. Because they were enriched with myofibroblasts as per the CiberSortx classification, we tentatively designated them as iCMS2-F/iCMS3-F. iCMS coupled with the application of an in silico cell fractionation method can provide the molecular dissection of CRC and adenoma.

Lung Cancer: A Literature Review

Lung cancer is a cancer whose onset starts in the lungs where there is an abnormal cell growth that is very fast and uncontrolled. The abnormal cell growth is triggered by deoxyribonucleic acid (DNA) damage, including deletions in the DNA section, inactivation of tumor suppressor genes, activation of proto-oncogenes to oncogenes, the absence of apoptosis, and the activity of the telomerase enzyme. Lung cancer is initiated by oncogeneous activity and inactivation of tumor suppressor genes. Oncogenes are genes that help cells grow and divide and are believed to cause a person to develop lung cancer. In general, lung cancer is divided into two types, namely non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). World Health Organization (WHO) classifies lung cancer based on histopathology into 4 major cell types, namely SCLC, NSCLC which includes adenocarcinoma, squamous cell carcinoma (SCC), and large cell carcinoma (LCC). The difference between the two is that SCLC has a higher aggressiveness than NSCLC. Cancer treatment is based on the type, size, location and stage of the cancer, as well as the patient's overall condition.

Taxifolin Inhibits the Growth of Non-Small-Cell Lung Cancer via Downregulating Genes Displaying Novel and Robust Associations with Immune Evasion Factors.

Using an LL2 cell-based syngeneic mouse LC model, taxifolin suppressed allografts along with the appearance of 578 differentially expressed genes (DEGs). These DEGs were associated with enhancement of processes related to the extracellular matrix and lymphocyte chemotaxis as well as the reduction in pathways relevant to cell proliferation. From these DEGs, we formulated 12-gene (TxflSig) and 7-gene (TxflSig1) panels; both predicted response to ICB (immune checkpoint blockade) therapy more effectively in non-small-cell lung cancer (NSCLC) than numerous well-established ICB biomarkers, including PD-L1. In both panels, the mouse counterparts of ITGAL, ITGAX, and TMEM119 genes were downregulated by taxifolin. They were strongly associated with immune suppression in LC, evidenced by their robust correlations with the major immunosuppressive cell types (MDSC, Treg, and macrophage) and multiple immune checkpoints in NSCLC and across multiple human cancer types. ITGAL, ITGAX, and IIT (ITGAL-ITGAX-TMEM119) effectively predicted NSCLC's response to ICB therapy; IIT stratified the mortality risk of NSCLC. The stromal expressions of ITGAL and ITGAX, together with tumor expression of TMEM119 in NSCLC, were demonstrated. Collectively, we report multiple novel ICB biomarkers-TxflSig, TxflSig1, IIT, ITGAL, and ITGAX-and taxifolin-derived attenuation of immunosuppressive activities in NSCLC, suggesting the inclusion of taxifolin in ICB therapies for NSCLC.

Investigation on tailoring the width and central frequency of bandgaps of TPMS structures

AbstractTriply periodic minimal surfaces (TPMS) lattice structures present outstanding properties such as lightweight, high strength, energy absorption, and wave propagation control, which are extensively investigated in recent years. However, one of the main challenges when designing TPMS is the proper selection of cell type and volume ratio in order to obtain the desired properties for specific applications. To this aim, this work provides a comprehensive numerical study of bandgap’s formation in the sub-2 kHz frequency range for the seven major cell type TPMS structures, including Primitive, Gyroid, Neovius, IWP, Diamond, Fischer–Koch S, and FRD, for a comprehensive range of volume ratios. Results show that these seven TPMS structures present a complete bandgap between the 3rd and 4th dispersion curves. The width of the bandgap is strongly dependent of the TPMS lattice and the widest bandgaps are seen on the Neovius and Primitive-based lattice (reaching a maximum width of 0.458 kHz and 0.483 kHz, respectively) for volume ratios over 0.3. Below this volume ratio, the bandgap of the Primitive structure becomes negligible, and the Neovius and IWP structures are the best candidates among the 7 tested TPMS cases. The central frequency of the bandgaps is less sensitive to the lattice and are predominantly tailored by the volume ratio. With this study, we demonstrate that the proper selection of the periodic cell type and volume ratio can tailor the bandwidth of complete bandgaps from a tens of Hz up to 0.48 kHz, while the central frequency can be selected from 0.72 to 1.81 kHz according to the volume ratio. The goal of this study is to serve as a database for the Primitive, Gyroid, Neovius, IWP, Diamond, Fischer–Koch S, and FRD TPMS structures for metamaterial designers.

Defining the Transcriptional Landscapes of the Tumor Microenvironment of Cervical and Vaginal Cancers at Single-Cell Resolution

Malignancies found within vaginal tissue are often diagnosed as cancers of the cervix, vulva, or urethra and are clinically treated with similar modalities. However, the rarity of vaginal cancer may be an artifice of categorization; current treatment paradigms do not take into account tissue-specific mutations and differences in mechanistic pathways intracellularly. Understanding the shared and distinctly different transcriptional profiles of vaginal and cervical tumors at a single-cell resolution will provide insights in vaginal tumor biology and will open avenues for future clinical interventions. Biopsies of tumor and adjacent normal tissue from 9 patients (3 adenocarcinomas (ADC), 3 squamous cell carcinomas (SCC) from the cervix, and 3 vaginal SCC) were collected and analyzed by single-cell RNA sequencing (scRNA-seq) to compare the tumor, immune, and stromal features of cervical and vaginal cancers. Collectively, over 50,000 cells were analyzed by scRNA-seq in this study. We performed dimensionality reduction and clustering analysis of the single-cell transcriptomes to identify the major cell types composing the vaginal and cervical tumor tissues. Compared to Cervical SCC, Vaginal SCC tissues showed reduced fractions of macrophages (-2.7 log2-fold; padj < 0.02) and T cells (-3.7 log2-fold; padj < 0.02) by differential cell proportion analysis (RAISIN). Likewise, the vaginal SCC epithelial cell compartments showed downregulation of inflammatory pathways including TNF signaling via NFKB (NES = -5.7, padj = 5.0 × 10-19), IL2 STAT5 signaling (NES = -4.5, padj = 1.6 × 10-12), and interferon gamma response (NES = -4.3, padj = 9.4 × 10-12), among the Hallmark pathway collection. On the other hand, vaginal SCC epithelial cells showed significant upregulation of oxidative phosphorylation (NES = 4.8, padj = 1.7 × 10-17), p53 pathway (NES = 4.2, padj = 1.8 × 10-13), mTORC1 signaling (NES = 4.2, padj = 1.9 × 10-13), and estrogen early and late response (NES = 4.0, padj < 7.5 × 10-12) compared to cervical SCC. These results highlight distinct differences in the cell type composition and cancer epithelial pathways in vaginal vs. cervical SCC. Among upregulated pathways in vaginal SCC, ER and mTORC1 pathway activation may represent targets for therapeutic intervention worthy of further investigation.

Astrocytes of the optic nerve exhibit a region-specific and temporally distinct response to elevated intraocular pressure

BackgroundThe optic nerve is an important tissue in glaucoma and the unmyelinated nerve head region remains an important site of many early neurodegenerative changes. In both humans and mice, astrocytes constitute the major glial cell type in the region, and in glaucoma they become reactive, influencing the optic nerve head (ONH) microenvironment and disease outcome. Despite recognizing their importance in the progression of the disease, the reactive response of optic nerve head astrocytes remains poorly understood.MethodsTo determine the global reactive response of ONH astrocytes in glaucoma we studied their transcriptional response to an elevation in IOP induced by the microbead occlusion model. To specifically isolate astrocyte mRNA in vivo from complex tissues, we used the ribotag method to genetically tag ribosomes in astrocytes, restricting analysis to astrocytes and enabling purification of astrocyte-associated mRNA throughout the entire cell, including the fine processes, for bulk RNA-sequencing. We also assessed the response of astrocytes in the more distal myelinated optic nerve proper (ONP) as glaucomatous changes manifest differently between the two regions.ResultsAstrocytes of the optic nerve exhibited a region-specific and temporally distinct response. Surprisingly, ONH astrocytes showed very few early transcriptional changes and ONP astrocytes demonstrated substantially larger changes over the course of the experimental period. Energy metabolism, particularly oxidative phosphorylation and mitochondrial protein translation emerged as highly upregulated processes in both ONH and ONP astrocytes, with the former showing additional upregulation in antioxidative capacity and proteolysis. Interestingly, optic nerve astrocytes demonstrated a limited neuroinflammatory response, even when challenged with a more severe elevation in IOP. Lastly, there were a greater number of downregulated processes in both astrocyte populations compared to upregulated processes.ConclusionOur findings demonstrate an essential role for energy metabolism in the response of optic nerve astrocytes to elevated IOP, and contrary to expectations, neuroinflammation had a limited overall role. The transcriptional response profile is supportive of the notion that optic nerve astrocytes have a beneficial role in glaucoma. These previously uncharacterized transcriptional response of optic nerve astrocytes to injury reveal their functional diversity and a greater heterogeneity than previously appreciated.

Genomic DNA activates the AIM2 inflammasome and STING pathways to induce inflammation in lacrimal gland myoepithelial cells

PurposePrimary Sjögren's syndrome (pSS) is an autoimmune disease that mainly attacks the lacrimal glands causing severe aqueous-deficient dry eye. Clinical evidence indicates the DNA sensing mechanism in the pathogenesis of pSS. The purpose of the present study is to determine the pro-inflammatory effect of self-genomic DNA (gDNA) on myoepithelial cells (MECs), which along with acinar and ductal cells is a major cell type of the lacrimal gland. MethodMECs primary culture was acquired from female C57BL6J mice. Genomic DNA was extracted from the spleen of the same animal. The MECs were challenged with self-gDNA. The cytokine secretion was detected using supernatant by enzyme-linked immunosorbent assay (ELISA). The activation of inflammasomes was determined using FAM-FLICA. Cryosections of NOD.B10.H2b mouse model of pSS were obtained for immunofluorescence microscopy (IF), with Balb/C as control. ResultTreatment with gDNA activated AIM2 inflammasome assembly and function, leading to secretion of interleukin (IL)-1β and IL-18 in MECs. The stimulation of IL-1β secretion by gDNA appeared to be solely at the post-translational level, whereas IL-18 secretion was a combination of increased protein synthesis and post-translational modification. Genomic DNA also induced the activation of STimulators of INterferon Genes (STING), which correlated to the activation of STING in the lacrimal gland from the NOD.B10.H2b mouse. STING activation led to the secretion of IFN-β via Nuclear Factor-κB (NF-κB). The IFN-β further enhances the secretion of IL-1β. The contractility of MECs was disabled by treatment with gDNA or poly AnT, independent of the level of intracellular [Ca2+]. ConclusionSelf-gDNA induces a proinflammatory response in lacrimal gland MECs by activating both the AIM2 inflammasome and STING and thus may contribute to the pathogenesis of pSS.

Anti-inflammatory and immunomodulating effects of rilpivirine: Relevance for the therapeutics of chronic liver disease

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease (CLD) worldwide and inflammation is key to its progression/resolution. As we have previously described that rilpivirine (RPV) is hepatoprotective in murine models of CLD, here we determine the molecular mechanisms involved, focusing on its anti-inflammatory and immunomodulating properties. They were evaluated in vitro (human hepatic cell lines of the major hepatic cell types), in vivo (liver samples from a murine nutritional model of NAFLD) and ex vivo (peripheral blood mononuclear cells -PBMC- from patients with CLD). Transcriptomic analysis of liver samples from NAFLD mice showed RPV down-regulated biological processes associated with the inflammatory response (NF-κB/IκB signaling and mitogen-activated protein kinase -MAPK- activity) and leukocyte chemotaxis and migration. We observed a decrease in Adgre1 and Ccr2 expression and in the number of CCR2 + cells in the periportal areas of RPV-treated NAFLD mice. This RPV-induced effect on the CCL2/CCR2 axis was confirmed in vitro. A similar result was also obtained with CXCL10/IP10, one of the main chemokines in the liver. RPV also diminished activation of MAP kinases p38 and JNK. In addition, RPV inhibited the NLRP3 inflammasome pathway in vitro, decreasing NLRP3 protein expression, caspase-1 activation and IL-1β gene expression. RPV was also proven anti-inflammatory in PBMC from patients with CLD treated ex vivo. In conclusion, beyond its well-described role in antiretroviral therapy, RPV manifests anti-inflammatory and immunoregulatory effects, a finding that could be of great relevance for the search of novel targets or repositioning strategies for CLD.

ScRNA-seq Reveals the Mechanism of Fatty Acid Desaturase 2 Mutation to Repress Leaf Growth in Peanut (Arachis hypogaea L.).

Fatty Acid Desaturase 2 (FAD2) controls the conversion of oleic acids into linoleic acids. Mutations in FAD2 not only increase the high-oleic content, but also repress the leaf growth. However, the mechanism by which FAD2 regulates the growth pathway has not been elucidated in peanut leaves with single-cell resolution. In this study, we isolated fad2 mutant leaf protoplast cells to perform single-cell RNA sequencing. Approximately 24,988 individual cells with 10,249 expressed genes were classified into five major cell types. A comparative analysis of 3495 differentially expressed genes (DEGs) in distinct cell types demonstrated that fad2 inhibited the expression of the cytokinin synthesis gene LOG in vascular cells, thereby repressing leaf growth. Further, pseudo-time trajectory analysis indicated that fad2 repressed leaf cell differentiation, and cell-cycle evidence displayed that fad2 perturbed the normal cell cycle to induce the majority of cells to drop into the S phase. Additionally, important transcription factors were filtered from the DEG profiles that connected the network involved in high-oleic acid accumulation (WRKY6), activated the hormone pathway (WRKY23, ERF109), and potentially regulated leaf growth (ERF6, MYB102, WRKY30). Collectively, our study describes different gene atlases in high-oleic and normal peanut seedling leaves, providing novel biological insights to elucidate the molecular mechanism of the high-oleic peanut-associated agronomic trait at the single-cell level.