Single-Cell RNA Sequencing Reveals the Spatial Heterogeneity and Functional Alteration of Endothelial Cells in Chronic Hepatitis B Infection.

Chronic Hepatitis B virus (CHB) infection is a global health challenge, causing damage ranging from hepatitis to cirrhosis and hepatocellular carcinoma. In our study, single-cell RNA sequencing (scRNA-seq) analysis was performed in livers from mice models with chronic inflammation induced by CHB infection and we found that endothelial cells (ECs) exhibited the largest number of differentially expressed genes (DEGs) among all ten cell types. NF-κB signaling was activated in ECs to induce cell dysfunction and subsequent hepatic inflammation, which might be mediated by the interaction of macrophage-derived and cholangiocyte-derived VISFATIN/Nampt signaling. Moreover, we divided ECs into three subclusters, including periportal ECs (EC_Z1), midzonal ECs (EC_Z2), and pericentral ECs (EC_Z3) according to hepatic zonation. Functional analysis suggested that pericentral ECs and midzonal ECs, instead of periportal ECs, were more vulnerable to HBV infection, as the VISFATIN/Nampt- NF-κB axis was mainly altered in these two subpopulations. Interestingly, pericentral ECs showed increasing communication with macrophages and cholangiocytes via the Nampt-Insr and Nampt-Itga5/Itgb1 axis upon CHB infection, which contribute to angiogenesis and vascular capillarization. Additionally, ECs, especially pericentral ECs, showed a close connection with nature killer (NK) cells and T cells via the Cxcl6-Cxcr6 axis, which is involved in shaping the microenvironment in CHB mice livers. Thus, our study described the heterogeneity and functional alterations of three subclusters in ECs. We revealed the potential role of VISFATIN/Nampt signaling in modulating ECs characteristics and related hepatic inflammation, and EC-derived chemokine Cxcl16 in shaping NK and T cell recruitment, providing key insights into the multifunctionality of ECs in CHB-associated pathologies.

Physical cell-cell contact elicits specific transcriptomic responses in wine yeast species.

Fermenting grape juice provides a habitat for a well-mapped and evolutionarily relevant microbial ecosystem consisting of many natural or inoculated strains of yeasts and bacteria. The molecular nature of many of the ecological interactions within this ecosystem remains poorly understood, with the partial exception of interactions of a metabolic nature such as competition for nutrients and production of toxic metabolites/peptides. Data suggest that physical contact between species plays a significant role in the phenotypic outcome of interspecies interactions. However, the molecular nature of the mechanisms regulating these phenotypes remains unknown. Here, we present a transcriptomic analysis of physical versus metabolic contact between two wine relevant yeast species, Saccharomyces cerevisiae and Lachancea thermotolerans. The data show that these species respond to the physical presence of the other species. In S. cerevisiae, physical contact results in the upregulation of genes involved in maintaining cell wall integrity, cell wall structural components, and genes involved in the production of H2S. In L. thermotolerans, HSP stress response genes were the most significantly upregulated gene family. Both yeasts downregulated genes belonging to the FLO family, some of which play prominent roles in cellular adhesion. qPCR analysis indicates that the expression of some of these genes is regulated in a species-specific manner, suggesting that yeasts adjust gene expression to specific biotic challenges or interspecies interactions. These findings provide fundamental insights into yeast interactions and evolutionary adaptations of these species to the wine ecosystem.IMPORTANCEWithin the wine ecosystem, yeasts are the most relevant contributors to alcoholic fermentation and wine organoleptic characteristics. While some studies have described yeast-yeast interactions during alcoholic fermentation, such interactions remain ill-defined, and little is understood regarding the molecular mechanisms behind many of the phenotypes observed when two or more species are co-cultured. In particular, no study has investigated transcriptional regulation in response to physical interspecies cell-cell contact, as opposed to the generally better understood/characterized metabolic interactions. These data are of direct relevance to our understanding of microbial ecological interactions in general while also creating opportunities to improve ecosystem-based biotechnological applications such as wine fermentation. Furthermore, the presence of competitor species has rarely been considered an evolutionary biotic selection pressure. In this context, the data reveal novel gene functions. This, and further such analysis, is likely to significantly enlarge the genome annotation space.

Single-cell and Bulk Transcriptomic Analyses Reveal a Stemness and Circadian Rhythm Disturbance-related Signature Predicting Clinical Outcome and Immunotherapy Response in Hepatocellular Carcinoma.

AIMS: Investigating the impact of stemness-related circadian rhythm disruption (SCRD) on hepatocellular carcinoma (HCC) prognosis and its potential as a predictor for immunotherapy response. BACKGROUND: Circadian disruption has been linked to tumor progression through its effect on the stemness of cancer cells. OBJECTIVE: Develop a novel signature for SCRD to accurately predict clinical outcomes and immune therapy response in patients with HCC. METHODS: The stemness degree of patients with HCC was assessed based on the stemness index (mRNAsi). The co-expression circadian genes significantly correlated with mRNAsi were identified and defined as stemness- and circadian-related genes (SCRGs). The SCRD scores of samples and cells were calculated based on the SCRGs. Differentially expressed genes with a prognostic value between distinct SCRD groups were identified in bulk and single-cell datasets to develop an SCRD signature. RESULTS: A higher SCRD score indicates a worse patient survival rate. Analysis of the tumor microenvironment revealed a significant correlation between SCRD and infiltrating immune cells. Heterogeneous expression patterns, functional states, genomic variants, and cell-cell interactions between two SCRD populations were revealed by transcriptomic, genomic, and interaction analyses. The robust SCRD signature for predicting immunotherapy response and prognosis in patients with HCC was developed and validated in multiple independent cohorts. CONCLUSIONS: In summary, distinct tumor immune microenvironment patterns were confirmed under SCRD in bulk and single-cell transcriptomic, and SCRD signature associated with clinical outcomes and immunotherapy response was developed and validated in HCC.

CACIMAR: cross-species analysis of cell identities, markers, regulations, and interactions using single-cell RNA sequencing data.

Transcriptomic analysis across species is increasingly used to reveal conserved gene regulations which implicate crucial regulators. Cross-species analysis of single-cell RNA sequencing (scRNA-seq) data provides new opportunities to identify the cellular and molecular conservations, especially for cell types and cell type-specific gene regulations. However, few methods have been developed to analyze cross-species scRNA-seq data to uncover both molecular and cellular conservations. Here, we built a tool called CACIMAR, which can perform cross-species analysis of cell identities, markers, regulations, and interactions using scRNA-seq profiles. Based on the weighted sum models of the conserved features, we developed different conservation scores to measure the conservation of cell types, regulatory networks, and intercellular interactions. Using publicly available scRNA-seq data on retinal regeneration in mice, zebrafish, and chick, we demonstrated four main functions of CACIMAR. First, CACIMAR allows to identify conserved cell types even in evolutionarily distant species. Second, the tool facilitates the identification of evolutionarily conserved or species-specific marker genes. Third, CACIMAR enables the identification of conserved intracellular regulations, including cell type-specific regulatory subnetworks and regulators. Lastly, CACIMAR provides a unique feature for identifying conserved intercellular interactions. Overall, CACIMAR facilitates the identification of evolutionarily conserved cell types, marker genes, intracellular regulations, and intercellular interactions, providing insights into the cellular and molecular mechanisms of species evolution.

Unveiling contact-mediated cellular crosstalk.

Cell-cell interactions orchestrate complex functions in multicellular organisms, forming a regulatory network for diverse biological processes. Their disruption leads to disease states. Recent advancements - including single-cell sequencing and spatial transcriptomics, coupled with powerful bioengineering and molecular tools - have revolutionized our understanding of how cells respond to each other. Notably, spatial transcriptomics allows us to analyze gene expression changes based on cell proximity, offering a unique window into the impact of cell-cell contact. Additionally, computational approaches are being developed to decipher how cell contact governs the symphony of cellular responses. This review explores these cutting-edge approaches, providing valuable insights into deciphering the intricate cellular changes influenced by cell-cell communication.

A Reference Profile of N-Glycosylation for Human Kidney and the Identification of Cell-Cell Interactions between Parietal Epithelial Cells and Capillary Endothelial Cells by Single-Cell Glycosylation-Sequencing.

BACKGROUND: N-glycosylation is one of the most common posttranslational modifications in humans, and these alterations are associated with kidney diseases. METHODS: A novel technological approach, single-cell N-acetyllactosamine sequencing (scLacNAc-seq), was applied to simultaneously detect N-glycosylation expression and the transcriptome at single-cell resolution in three human kidney tissues from zero-time biopsy. Cell clusters, glycation abundance in each cell cluster, functional enrichment analysis, cell-cell crosstalk, and pseudotime analysis were applied. RESULTS: Using scLacNAc-seq, 24,247 cells and 22 cell clusters were identified, and N-glycan abundance in each cell was obtained. Transcriptome analysis revealed a close connection between capillary endothelial cells (CapECs) and parietal epithelial cells (PECs). PECs and CapECs communicate with each other through several pairs of ligand receptors (e.g., TGFB1-EGFR, GRN-EGFR, TIMP1-FGFR2, VEGFB-FLT1, ANGPT2-TEK, and GRN-TNFRSF1A). Finally, a regulatory network of cell-cell crosstalk between PECs and CapECs was constructed, which is involved in cell development. CONCLUSIONS: We here, for the first time, constructed the glycosylation profile of 22 cell clusters in the human kidney from zero-time biopsy. Moreover, cell-cell communication between PECs and CapECs through the ligand-receptor system may play a crucial regulatory role in cell proliferation.

Making Ramón y Cajal proud: Development of cell identity and diversity in the cerebral cortex.

Since the beautiful images of Santiago Ramón y Cajal provided a first glimpse into the immense diversity and complexity of cell types found in the cerebral cortex, neuroscience has been challenged and inspired to understand how these diverse cells are generated and how they interact with each other to orchestrate the development of this remarkable tissue. Some fundamental questions drive the field's quest to understand cortical development: what are the mechanistic principles that govern the emergence of neuronal diversity? How do extrinsic and intrinsic signals integrate with physical forces and activity to shape cell identity? How do the diverse populations of neurons and glia influence each other during development to guarantee proper integration and function? The advent of powerful new technologies to profile and perturb cortical development at unprecedented resolution and across a variety of modalities has offered a new opportunity to integrate past knowledge with brand new data. Here, we review some of this progress using cortical excitatory projection neurons as a system to draw out general principles of cell diversification and the role of cell-cell interactions during cortical development.

Transcript and protein signatures derived from shared molecular interactions across cancers are associated with mortality.

BACKGROUND: Characterization of shared cancer mechanisms have been proposed to improve therapy strategies and prognosis. Here, we aimed to identify shared cell-cell interactions (CCIs) within the tumor microenvironment across multiple solid cancers and assess their association with cancer mortality. METHODS: CCIs of each cancer were identified by NicheNet analysis of single-cell RNA sequencing data from breast, colon, liver, lung, and ovarian cancers. These CCIs were used to construct a shared multi-cellular tumor model (shared-MCTM) representing common CCIs across cancers. A gene signature was identified from the shared-MCTM and tested on the mRNA and protein level in two large independent cohorts: The Cancer Genome Atlas (TCGA, 9185 tumor samples and 727 controls across 22 cancers) and UK biobank (UKBB, 10,384 cancer patients and 5063 controls with proteomics data across 17 cancers). Cox proportional hazards models were used to evaluate the association of the signature with 10-year all-cause mortality, including sex-specific analysis. RESULTS: A shared-MCTM was derived from five individual cancers. A shared gene signature was extracted from this shared-MCTM and the most prominent regulatory cell type, matrix cancer-associated fibroblast (mCAF). The signature exhibited significant expression changes in multiple cancers compared to controls at both mRNA and protein levels in two independent cohorts. Importantly, it was significantly associated with mortality in cancer patients in both cohorts. The highest hazard ratios were observed for brain cancer in TCGA (HR [95%CI] = 6.90[4.64-10.25]) and ovarian cancer in UKBB (5.53[2.08-8.80]). Sex-specific analysis revealed distinct risks, with a higher mortality risk associated with the protein signature score in males (2.41[1.97-2.96]) compared to females (1.84[1.44-2.37]). CONCLUSION: We identified a gene signature from a comprehensive shared-MCTM representing common CCIs across different cancers and revealed the regulatory role of mCAF in the tumor microenvironment. The pathogenic relevance of the gene signature was supported by differential expression and association with mortality on both mRNA and protein levels in two independent cohorts.

Myocardial B cells have specific gene expression and predicted interactions in dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy.

Introduction: Growing evidence from animal models indicates that the myocardium hosts a population of B cells that play a role in the development of cardiomyopathy. However, there is minimal data on human myocardial B cells in the context of cardiomyopathy. Methods: We integrated single-cell and single-nuclei datasets from 45 healthy human hearts, 70 hearts with dilated cardiomyopathy (DCM), and 8 hearts with arrhythmogenic right ventricular cardiomyopathy (ARVC). Interactions between B cells and other cell types were investigated using the CellChat Package. Differential gene expression analysis comparing B cells across conditions was performed using DESeq2. Pathway analysis was performed using Ingenuity, KEGG, and GO pathways analysis. Results: We identified 1,100 B cells, including naive B cells and plasma cells. Cells showed an extensive network of interactions within the healthy myocardium that included outgoing signaling to macrophages, T cells, endothelial cells, and pericytes, and incoming signaling from endothelial cells, pericytes, and fibroblasts. This niche relied on ECM-receptor, contact, and paracrine interactions; and changed significantly in the context of cardiomyopathy, displaying disease-specific features. Differential gene expression analysis showed that in the context of DCM both naive and plasma B cells upregulated several pathways related to immune activation, including upregulation of oxidative phosphorylation, upregulation of leukocyte extravasation, and, in naive B cells, antigen presentation. Discussion: The human myocardium contains naive B cells and plasma cells, integrated into a diverse and dynamic niche that has distinctive features in healthy, DCM, and ARVC. Naive myocardial-associated B cells likely contribute to the pathogenesis of human DCM.

Computational analysis of super-resolved in situ sequencing data reveals genes modified by immune-tumor contact events.

Cancer cells can manipulate immune cells and escape from the immune system response. Quantifying the molecular changes that occur when an immune cell touches a tumor cell can increase our understanding of the underlying mechanisms. Recently, it became possible to perform such measurements in situ-for example, using expansion sequencing, which enabled in situ sequencing of genes with super-resolution. We systematically examined whether individual immune cells from specific cell types express genes differently when in physical proximity to individual tumor cells. First, we demonstrated that a dense mapping of genes in situ can be used for the segmentation of cell bodies in 3D, thus improving our ability to detect likely touching cells. Next, we used three different computational approaches to detect the molecular changes that are triggered by proximity: differential expression analysis, tree-based machine learning classifiers, and matrix factorization analysis. This systematic analysis revealed tens of genes, in specific cell types, whose expression separates immune cells that are proximal to tumor cells from those that are not proximal, with a significant overlap between the different detection methods. Remarkably, an order of magnitude more genes are triggered by proximity to tumor cells in CD8 T cells compared to CD4 T cells, in line with the ability of CD8 T cells to directly bind major histocompatibility complex (MHC) class I on tumor cells. Thus, in situ sequencing of an individual biopsy can be used to detect genes likely involved in immune-tumor cell-cell interactions. The data used in this manuscript and the code of the InSituSeg, machine learning, cNMF, and Moran's I methods are publicly available at doi:10.5281/zenodo.7497981.

N-cadherin directs the collective Schwann cell migration required for nerve regeneration through Slit2/3-mediated contact inhibition of locomotion.

Collective cell migration is fundamental for the development of organisms and in the adult for tissue regeneration and in pathological conditions such as cancer. Migration as a coherent group requires the maintenance of cell-cell interactions, while contact inhibition of locomotion (CIL), a local repulsive force, can propel the group forward. Here we show that the cell-cell interaction molecule, N-cadherin, regulates both adhesion and repulsion processes during Schwann cell (SC) collective migration, which is required for peripheral nerve regeneration. However, distinct from its role in cell-cell adhesion, the repulsion process is independent of N-cadherin trans-homodimerisation and the associated adherens junction complex. Rather, the extracellular domain of N-cadherin is required to present the repulsive Slit2/Slit3 signal at the cell surface. Inhibiting Slit2/Slit3 signalling inhibits CIL and subsequently collective SC migration, resulting in adherent, nonmigratory cell clusters. Moreover, analysis of ex vivo explants from mice following sciatic nerve injury showed that inhibition of Slit2 decreased SC collective migration and increased clustering of SCs within the nerve bridge. These findings provide insight into how opposing signals can mediate collective cell migration and how CIL pathways are promising targets for inhibiting pathological cell migration.

Investigating the paracrine and juxtacrine abilities of adipose-derived stromal cells in angiogenesis triple cell co-cultures.

The pro-angiogenic abilities of adipose-derived stromal cells (ASCs) make them attractive candidates for cellular therapy, especially for ischemic disease indications. However, details regarding the underlying mechanisms remain elusive. Therefore, this study aimed to investigate paracrine and juxtacrine abilities of ASCs in angiogenesis triple cell co-cultures by detailed image analysis of the vascular-like structures. Fibroblast-endothelial cell co-cultures were established, and ASCs were added directly or indirectly through inserts. The cultures were treated with antibodies or subjected to analyses using ELISA and RT2 PCR Arrays. The model consistently generated vascular-like structures. ASCs increased the total branch lengths equally well in paracrine and juxtacrine conditions, by increasing the number of branches and average branch lengths (ABL). In contrast, addition of VEGF to the model increased the number of branches, but not the ABL. Still, ASCs increased the VEGF levels in supernatants of paracrine and juxtacrine co-cultures, and anti-VEGF treatment decreased the sprouting. ASCs themselves up-regulated collagen type V in response to paracrine signals from the co-cultures. The results suggest that ASCs initiate sprouting through secretion of several paracrine factors, among which VEGF is identified, but VEGF alone does not recapitulate the paracrine actions of ASCs. By employing neutralizing antibodies and dismantling common model outputs using image analysis, the triple cell co-culture is an attractive tool for discovery of the paracrine factors in ASCs' secretome which act in concert with VEGF to improve angiogenesis.

Inter-plane feedback coordinates cell morphogenesis and maintains 3D tissue organization in the Drosophila pupal retina.

How complex organs coordinate cellular morphogenetic events to achieve three-dimensional (3D) form is a central question in development. The question is uniquely tractable in the late Drosophila pupal retina, where cells maintain stereotyped contacts as they elaborate the specialized cytoskeletal structures that pattern the apical, basal and longitudinal planes of the epithelium. In this study, we combined cell type-specific genetic manipulation of the cytoskeletal regulator Abelson (Abl) with 3D imaging to explore how the distinct cellular morphogenetic programs of photoreceptors and interommatidial pigment cells (IOPCs) organize tissue pattern to support retinal integrity. Our experiments show that photoreceptor and IOPC terminal differentiation is unexpectedly interdependent, connected by an intercellular feedback mechanism that coordinates and promotes morphogenetic change across orthogonal tissue planes to ensure correct 3D retinal pattern. We propose that genetic regulation of specialized cellular differentiation programs combined with inter-plane mechanical feedback confers spatial coordination to achieve robust 3D tissue morphogenesis.

Isolation and draft genome sequence of Enterobacter asburiae strain i6 amenable to genetic manipulation.

Enterobacter asburiae is a species of Gram-negative bacteria that is found in soil, water, and sewage. E. asburiae is generally considered to be an opportunistic pathogen, but has also been reported as a plant growth-promoting bacterium (PGPB), which may have beneficial effects on plant growth and development. However, genetic analysis of E. asburiae has been limited, possibly due to its redundant enzymes that digest exogenous DNA in the cell. Here, an E. asburiae strain i6 was isolated from soil in Nara, Japan. This strain was amenable to transformation and the one-step gene inactivation method based on λ Red recombinase. The transformation efficiency of the i6 strain with the 10 kb plasmid DNA pCF430 was at least four orders of magnitude higher than that of the previously sequenced E. asburiae strain ATCC 35953, which could not be transformed with the same plasmid DNA. A draft genome sequence of the i6 strain was determined and deposited into the database, allowing several factors that may determine transformation efficiency to be perturbed and tested. Together with the amenability of the i6 strain to genetic manipulation, the information from the i6 genome will facilitate characterization and fine-tuning of the beneficial and detrimental traits of this species.

Collective durotaxis along a self-generated mobile stiffness gradient in vivo.

A crucial aspect of tissue self-organization during morphogenesis, wound healing, and cancer invasion is directed migration of cell collectives. The majority of in vivo directed migration has been guided by chemotaxis, whereby cells follow a chemical gradient. In certain situations, migrating cell collectives can also self-generate the stiffness gradient in the surrounding tissue, which can have a feedback effect on the directionality of the migration. The phenomenon has been observed during collective durotaxis in vivo. Along the biointerface between neighbouring tissues, heterotypic cell-cell interactions are the main cause of this self-generated stiffness gradient. The physical processes in charge of tissue self-organization along the biointerface, which are related to the interplay between cell signalling and the formation of heterotypic cell-cell adhesion contacts, are less well-developed than the biological mechanisms of the cellular interactions. This complex phenomenon is discussed here in the model system, such as collective migration of neural crest cells between ectodermal placode and mesoderm subpopulations within Xenopus embryos by pointing to the role of the dynamics along the biointerface between adjacent cell subpopulations on the subpopulation stiffness.

Enabling neighbour labelling: using synthetic biology to explore how cells influence their neighbours.

Cell-cell interactions are central to development, but exploring how a change in any given cell relates to changes in the neighbour of that cell can be technically challenging. Here, we review recent developments in synthetic biology and image analysis that are helping overcome this problem. We highlight the opportunities presented by these advances and discuss opportunities and limitations in applying them to developmental model systems.

Immunomodulatory differences between mesenchymal stem cells from different oral tissues.

Mesenchymal stem cells (MSCs) have recently been identified as having potentially therapeutic immunomodulatory properties. MSCs isolated from different oral tissues have similar morphology and immunophenotypes, however, direct comparisons of their gene expression and immunomodulatory properties have not been conducted. We isolated alveolar bone-derived MSCs (aBMSCs), dental pulp stem cells (DPSCs) and gingiva-derived MSCs (GMSCs) from the same patients and compared their immunophenotypes and transcriptomes. Additionally, we compared their production of soluble immunomodulatory cytokines as well as their immunoregulatory properties in coculture with THP-1 human monocytic cells. RNA sequencing revealed distinct gene expression in DPSCs while aBMSCs and GMSCs had less differentially expressed genes. DPSCs also had significantly less secretion of osteopontin compared to aBMSCs and GMSCs. Finally, DPSCs did not exhibit an immunosuppresive effect on THP-1 cells to the same degree as aBMSCs and GMSCs. These findings demonstrate that MSCs from different oral tissues have distinct transcriptomes and immunoregulatory properties.

The free energy landscape of small-world networks of cells.

The properties of organs, tissues, organoids, and other systems of cells, are influenced by the spatial localization and distribution of their elements. Here, we used networks to describe distributions of cells on a surface where the small-world coefficient (SW) of the networks was varied between SW~1 (random uniform distributions) and SW~10 (clustered distributions). The small-world coefficient is a topological measure of graphs: networks with SW>1 are topologically biased to transmit information. For each system configuration, we then determined the total energy U as the sum of the energies that describe cell-cell interactions - approximated by a harmonic potential. The graph of energy (U) across the configuration space of the networks (SW) is the energy landscape: it indicates which configuration a system of cells will likely assume over time. We found that, depending on the model parameters, the energy landscapes of 2D distributions of cells may be of different types: from type I to type IV. Type I and type II systems have high probability to evolve into random distributions. Type III and type IV systems have a higher probability to form clustered architectures. A great many of simulations indicated that cultures of cells with high initial density and limited sensing range could evolve into clustered configurations with enhanced topological characteristics. Moreover, the strongest the binding between cells, the greater the likelihood that they will assume configurations characterized by finite values of SW. Results of the work are relevant for those working the field of tissue engineering, regenerative medicine, the formation of in-vitro-models, the analysis of neuro-degenerative diseases.