Single-cell transcriptomic sequencing analysis of mechanistic insights into the IFN-γ signaling pathway in different tumor cells.

PURPOSE: This study aimed to investigate the relationship between the interferon-gamma (IFN-γ) pathway in different tumor microenvironments (TME) and patients' prognosis, as well as the regulatory mechanisms of this pathway in tumor cells. METHODS: Using RNA-seq data from the TCGA database, we analyzed the predictive value of the IFN-γ pathway across various tumors. We employed a univariate Cox regression model to assess the prognostic significance of IFN-γ signaling in different tumor types. Additionally, we analyzed single-cell RNA sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) database to examine the distribution characteristics of the IFN-γ pathway and explore its regulatory mechanisms, highlighting how IFN-γ influenced cellular interactions within the TME. RESULTS: Our analysis revealed a significant association between the IFN-γ pathway and adverse prognosis in pan-cancer tissues (P < 0.001). Interestingly, this correlation varied regarding positive and negative regulation across different tumor types. Through a detailed examination of scRNA-seq data, we found that the IFN-γ pathway exerted substantial regulatory effects on stromal and immune cells. In contrast, its expression and regulatory patterns in tumor cells exhibited diversity and heterogeneity. Further analysis indicated that the IFN-γ pathway not only enhanced the immunogenicity of tumor cells but also inhibited their proliferation. Cell-cell interaction analysis confirmed the pivotal role of the IFN-γ pathway within the overall regulatory network. Moreover, we identified HMGB2 (high mobility group box 2) in T cells as a potential key regulator of tumor cell proliferation. CONCLUSIONS: The IFN-γ pathway exhibited a dual function by both suppressing tumor cell proliferation and enhancing their immunogenicity, positioning it as a pivotal target for refined cancer diagnosis and cancer strategies.

Diffusion on PCA-UMAP Manifold: The Impact of Data Structure Preservation to Denoise High-Dimensional Single-Cell RNA Sequencing Data.

Single-cell transcriptomics (scRNA-seq) is revolutionizing biological research, yet it faces challenges such as inefficient transcript capture and noise. To address these challenges, methods like neighbor averaging or graph diffusion are used. These methods often rely on k-nearest neighbor graphs from low-dimensional manifolds. However, scRNA-seq data suffer from the 'curse of dimensionality', leading to the over-smoothing of data when using imputation methods. To overcome this, sc-PHENIX employs a PCA-UMAP diffusion method, which enhances the preservation of data structures and allows for a refined use of PCA dimensions and diffusion parameters (e.g., k-nearest neighbors, exponentiation of the Markov matrix) to minimize noise introduction. This approach enables a more accurate construction of the exponentiated Markov matrix (cell neighborhood graph), surpassing methods like MAGIC. sc-PHENIX significantly mitigates over-smoothing, as validated through various scRNA-seq datasets, demonstrating improved cell phenotype representation. Applied to a multicellular tumor spheroid dataset, sc-PHENIX identified known extreme phenotype states, showcasing its effectiveness. sc-PHENIX is open-source and available for use and modification.

Neuronal identity control at the resolution of a single transcription factor isoform.

The brain exhibits remarkable neuronal diversity which is critical for its functional integrity. From the sheer number of cell types emerging from extensive transcriptional, morphological, and connectome datasets, the question arises of how the brain is capable of generating so many unique identities. 'Terminal selectors' are transcription factors hypothesized to determine the final identity characteristics in post-mitotic cells. Which transcription factors function as terminal selectors and the level of control they exert over different terminal characteristics are not well defined. Here, we establish a novel role for the transcription factor broad as a terminal selector in Drosophila melanogaster. We capitalize on existing large sequencing and connectomics datasets and employ a comprehensive characterization of terminal characteristics including Perturb-seq and whole-cell electrophysiology. We find a single isoform broad-z4 serves as the switch between the identity of two visual projection neurons LPLC1 and LPLC2. Broad-z4 is natively expressed in LPLC1, and is capable of transforming the transcriptome, morphology, and functional connectivity of LPLC2 cells into LPLC1 cells when perturbed. Our comprehensive work establishes a single isoform as the smallest unit underlying an identity switch, which may serve as a conserved strategy replicated across developmental programs.

Data normalization for addressing the challenges in the analysis of single-cell transcriptomic datasets.

BACKGROUND: Normalization is a critical step in the analysis of single-cell RNA-sequencing (scRNA-seq) datasets. Its main goal is to make gene counts comparable within and between cells. To do so, normalization methods must account for technical and biological variability. Numerous normalization methods have been developed addressing different sources of dispersion and making specific assumptions about the count data. MAIN BODY: The selection of a normalization method has a direct impact on downstream analysis, for example differential gene expression and cluster identification. Thus, the objective of this review is to guide the reader in making an informed decision on the most appropriate normalization method to use. To this aim, we first give an overview of the different single cell sequencing platforms and methods commonly used including isolation and library preparation protocols. Next, we discuss the inherent sources of variability of scRNA-seq datasets. We describe the categories of normalization methods and include examples of each. We also delineate imputation and batch-effect correction methods. Furthermore, we describe data-driven metrics commonly used to evaluate the performance of normalization methods. We also discuss common scRNA-seq methods and toolkits used for integrated data analysis. CONCLUSIONS: According to the correction performed, normalization methods can be broadly classified as within and between-sample algorithms. Moreover, with respect to the mathematical model used, normalization methods can further be classified into: global scaling methods, generalized linear models, mixed methods, and machine learning-based methods. Each of these methods depict pros and cons and make different statistical assumptions. However, there is no better performing normalization method. Instead, metrics such as silhouette width, K-nearest neighbor batch-effect test, or Highly Variable Genes are recommended to assess the performance of normalization methods.

Genomics, Transcriptomics, and Epigenetics of Sporadic Pituitary Tumors.

Pituitary tumors (PT) are highly heterogeneous neoplasms, comprising functioning and nonfunctioning lesions. Functioning PT include prolactinomas, causing amenorrhea-galactorrhea in women and sexual dysfunction in men; GH-secreting adenomas causing acromegaly-gigantism; ACTH-secreting corticotrophinomas causing Cushing disease (CD); and the rare TSH-secreting thyrotrophinomas that result in central hyperthyroidism. Nonfunctioning PT do not result in a hormonal hypersecretion syndrome and most of them are of gonadotrope differentiation; other non-functioning PT include null cell adenomas and silent ACTH-, GH- and PRL-adenomas. Less than 5% of PT occur in a familial or syndromic context whereby germline mutations of specific genes account for their molecular pathogenesis. In contrast, the more common sporadic PT do not result from a single molecular abnormality but rather emerge from several oncogenic events that culminate in an increased proliferation of pituitary cells, and in the case of functioning tumors, in a non-regulated hormonal hypersecretion. In recent years, important advances in the understanding of the molecular pathogenesis of PT have been made, including the genomic, transcriptomic, epigenetic, and proteomic characterization of these neoplasms. In this review, we summarize the available molecular information pertaining the oncogenesis of PT.

Machine Learning and COVID-19: Lessons from SARS-CoV-2.

Currently, methods in machine learning have opened a significant number of applications to construct classifiers with capacities to recognize, identify, and interpret patterns hidden in massive amounts of data. This technology has been used to solve a variety of social and health issues against coronavirus disease 2019 (COVID-19). In this chapter, we present some supervised and unsupervised machine learning techniques that have contributed in three aspects to supplying information to health authorities and diminishing the deadly effects of the current worldwide outbreak on the population. First is the identification and construction of powerful classifiers capable of predicting severe, moderate, or asymptomatic responses in COVID-19 patients starting from clinical or high-throughput technologies. Second is the identification of groups of patients with similar physiological responses to improve the triage classification and inform treatments. The final aspect is the combination of machine learning methods and schemes from systems biology to link associative studies with mechanistic frameworks. This chapter aims to discuss some practical applications in the use of machine learning techniques to handle data coming from social behavior and high-throughput technologies, associated with COVID-19 evolution.

Podoplanin and CLEC-2 levels in patients with COVID-19.

Introduction: Podoplanin (PDPN gene) and CLEC-2 are involved in inflammatory hemostasis and have also been related with the pathogenesis of thrombosis. Emerging evidence also suggest that podoplanin can exert protective effects in sepsis and in acute lung injury. In lungs, podoplanin is co-expressed with ACE2, which is the main entry receptor for SARS-CoV-2. Aim: To explore the role of podoplanin and CLEC-2 in COVID-19. Methods: Circulating levels of podoplanin and CLEC-2 were measured in 30 consecutive COVID-19 patients admitted due to hypoxia, and in 30 age- and sex-matched healthy individuals. Podoplanin expression in lungs from patients who died of COVID-19 was obtained from two independent public databases of single-cell RNAseq from which data from control lungs were also available. Results: Circulating podoplanin levels were lower in COVID-19, while no difference was observed in CLEC-2 levels. Podoplanin levels were significantly inversely correlated with markers of coagulation, fibrinolysis and innate immunity. scRNAseq data confirmed that PDPN is co-expressed with ACE2 in pneumocytes, and showed that PDPN expression is lower in this cell compartment in lungs from patients with COVID-19. Conclusion: Circulating levels of podoplanin are lower in COVID-19, and the magnitude of this reduction is correlated with hemostasis activation. We also demonstrate the downregulation of PDPN at the transcription level in pneumocytes. Together, our exploratory study questions whether an acquired podoplanin deficiency could be involved in the pathogenesis of acute lung injury in COVID-19, and warrant additional studies to confirm and refine these findings.

Cell annotation using scRNA-seq data: A protein-protein interaction network approach.

Pathway analysis is an important step in the interpretation of single cell transcriptomic data, as it provides powerful information to detect which cellular processes are active in each individual cell. We have recently developed a protein-protein interaction network-based framework to quantify pluripotency associated pathways from scRNA-seq data. On this occasion, we extend this approach to quantify the activity of a pathway associated with any biological process, or even any list of genes. A systems-level characterization of pathway activities across multiple cell types provides a broadly applicable tool for the analysis of pathways in both healthy and disease conditions. Dysregulated cellular functions are a hallmark of a wide spectrum of human disorders, including cancer and autoimmune diseases. Here, we illustrate our method by analyzing various biological processes in healthy and cancer breast samples. Using this approach we found that tumor breast cells, even when they form a single group in the UMAP space, keep diverse biological programs active in a differentiated manner within the cluster.•We implement a protein-protein interaction network-based approach to quantify the activity of different biological processes.•The methodology can be used for cell annotation in scRNA-seq studies and is freely available as R package.

Machine learning applications on intratumoral heterogeneity in glioblastoma using single-cell RNA sequencing data.

Artificial intelligence is revolutionizing all fields that affect people's lives and health. One of the most critical applications is in the study of tumors. It is the case of glioblastoma (GBM) that has behaviors that need to be understood to develop effective therapies. Due to advances in single-cell RNA sequencing (scRNA-seq), it is possible to understand the cellular and molecular heterogeneity in the GBM. Given that there are different cell groups in these tumors, there is a need to apply Machine Learning (ML) algorithms. It will allow extracting information to understand how cancer changes and broaden the search for effective treatments. We proposed multiple comparisons of ML algorithms to classify cell groups based on the GBM scRNA-seq data. This broad comparison spectrum can show the scientific-medical community which models can achieve the best performance in this task. In this work are classified the following cell groups: Tumor Core (TC), Tumor Periphery (TP) and Normal Periphery (NP), in binary and multi-class scenarios. This work presents the biomarker candidates found for the models with the best results. The analyses presented here allow us to verify the biomarker candidates to understand the genetic characteristics of GBM, which may be affected by a suitable identification of GBM heterogeneity. This work obtained for the four scenarios covered cross-validation results of $93.03\% \pm 5.37\%$, $97.42\% \pm 3.94\%$, $98.27\% \pm 1.81\%$ and $93.04\% \pm 6.88\%$ for the classification of TP versus TC, TP versus NP, NP versus TP and TC (TPC) and NP versus TP versus TC, respectively.

Spermiogenesis alterations in the absence of CTCF revealed by single cell RNA sequencing.

CTCF is an architectonic protein that organizes the genome inside the nucleus in almost all eukaryotic cells. There is evidence that CTCF plays a critical role during spermatogenesis as its depletion produces abnormal sperm and infertility. However, defects produced by its depletion throughout spermatogenesis have not been fully characterized. In this work, we performed single cell RNA sequencing in spermatogenic cells with and without CTCF. We uncovered defects in transcriptional programs that explain the severity of the damage in the produced sperm. In the early stages of spermatogenesis, transcriptional alterations are mild. As germ cells go through the specialization stage or spermiogenesis, transcriptional profiles become more altered. We found morphology defects in spermatids that support the alterations in their transcriptional profiles. Altogether, our study sheds light on the contribution of CTCF to the phenotype of male gametes and provides a fundamental description of its role at different stages of spermiogenesis.

Bioinformatic analysis of single-cell RNA sequencing dataset dissects cellular heterogeneity of triple-negative breast cancer in transcriptional profile, splicing event and crosstalk network.

BACKGROUND: Triple-negative breast cancer (TNBC) is a subtype of breast cancer with high tumoral heterogeneity, while the detailed regulatory network is not well known. METHODS: Via single-cell RNA-sequencing (scRNA-seq) data analysis, we comprehensively investigated the transcriptional profile of different subtypes of TNBC epithelial cells with gene regulatory network (GRN) and alternative splicing (AS) event analysis, as well as the crosstalk between epithelial and non-epithelial cells. RESULTS: Of note, we found that luminal progenitor subtype exhibited the most complex GRN and splicing events. Besides, hnRNPs negatively regulates AS events in luminal progenitor subtype. In addition, we explored the cellular crosstalk among endothelial cells, stromal cells and immune cells in TNBC and discovered that NOTCH4 was a key receptor and prognostic marker in endothelial cells, which provide potential biomarker and target for TNBC intervention. CONCLUSIONS: In summary, our study elaborates on the cellular heterogeneity of TNBC, revealing that NOTCH4 in endothelial cells was critical for TNBC intervention. This in-depth understanding of epithelial cell and non-epithelial cell network would provide theoretical basis for the development of new drugs targeting this sophisticated network in TNBC.

ORIGINS: A protein network-based approach to quantify cell pluripotency from scRNA-seq data.

Trajectory inference is a common application of scRNA-seq data. However, it is often necessary to previously determine the origin of the trajectories, the stem or progenitor cells. In this work, we propose a computational tool to quantify pluripotency from single cell transcriptomics data. This approach uses the protein-protein interaction (PPI) network associated with the differentiation process as a scaffold and the gene expression matrix to calculate a score that we call differentiation activity. This score reflects how active the differentiation network is in each cell. We benchmark the performance of our algorithm with two previously published tools, LandSCENT (Chen et al., 2019) and CytoTRACE (Gulati et al., 2020), for four healthy human data sets: breast, colon, hematopoietic and lung. We show that our algorithm is more efficient than LandSCENT and requires less RAM memory than the other programs. We also illustrate a complete workflow from the count matrix to trajectory inference using the breast data set.•ORIGINS is a methodology to quantify pluripotency from scRNA-seq data implemented as a freely available R package.•ORIGINS uses the protein-protein interaction network associated with differentiation and the data set expression matrix to calculate a score (differentiation activity) that quantifies pluripotency for each cell.

Single-cell transcriptomics reveals hidden information in trypanosomatids.

Dixenic parasites often encounter environmental extremes during the transition from vector to host. Preadapted transmission stages overcome these challenges to promote parasites' survival and ensure life cycle progression. Recently, Vigneron et al. and Briggs et al. used single-cell transcriptomics to investigate developmental stage specific gene expression patterns during parasite differentiation.

An Integrative Single-cell Transcriptomic Atlas of the Post-natal Mouse Mammary Gland Allows Discovery of New Developmental Trajectories in the Luminal Compartment.

The mammary gland is a highly dynamic organ which undergoes periods of expansion, differentiation and cell death in each reproductive cycle. Partly because of the dynamic nature of the gland, mammary epithelial cells (MECs) are extraordinarily heterogeneous. Single cell RNA-seq (scRNA-seq) analyses have contributed to understand the cellular and transcriptional heterogeneity of this complex tissue. Here, we integrate scRNA-seq data from three foundational reports that have explored the mammary gland cell populations throughout development at single-cell level using 10× Chromium Drop-Seq. We center our analysis on post-natal development of the mammary gland, from puberty to post-involution. The new integrated study corresponds to RNA sequences from 53,686 individual cells, which greatly outnumbers the three initial data sets. The large volume of information provides new insights, as a better resolution of the previously detected Procr+ stem-like cell subpopulation or the identification of a novel group of MECs expressing immune-like markers. Moreover, here we present new pseudo-temporal trajectories of MEC populations at two resolution levels, that is either considering all mammary cell subtypes or focusing specifically on the luminal lineages. Interestingly, the luminal-restricted analysis reveals distinct expression patterns of various genes that encode milk proteins, suggesting specific and non-redundant roles for each of them. In summary, our data show that the application of bioinformatic tools to integrate multiple scRNA-seq data-sets helps to describe and interpret the high level of plasticity involved in gene expression regulation throughout mammary gland post-natal development.

Increased expression of hypoxia-induced factor 1α mRNA and its related genes in myeloid blood cells from critically ill COVID-19 patients.

BACKGROUND: COVID-19 counts 46 million people infected and killed more than 1.2 million. Hypoxaemia is one of the main clinical manifestations, especially in severe cases. HIF1α is a master transcription factor involved in the cellular response to oxygen levels. The immunopathogenesis of this severe form of COVID-19 is poorly understood. METHODS: We performed scRNAseq from leukocytes from five critically ill COVID-19 patients and characterized the expression of hypoxia-inducible factor1α and its transcriptionally regulated genes. Also performed metanalysis from the publicly available RNAseq data from COVID-19 bronchoalveolar cells. RESULTS: Critically-ill COVID-19 patients show a shift towards an immature myeloid profile in peripheral blood cells, including band neutrophils, immature monocytes, metamyelocytes, monocyte-macrophages, monocytoid precursors, and promyelocytes-myelocytes, together with mature monocytes and segmented neutrophils. May be the result of a physiological response known as emergency myelopoiesis. These cellular subsets and bronchoalveolar cells express HIF1α and their transcriptional targets related to inflammation (CXCL8, CXCR1, CXCR2, and CXCR4); virus sensing, (TLR2 and TLR4); and metabolism (SLC2A3, PFKFB3, PGK1, GAPDH and SOD2). CONCLUSIONS: The up-regulation and participation of HIF1α in events such as inflammation, immunometabolism, and TLR make it a potential molecular marker for COVID-19 severity and, interestingly, could represent a potential target for molecular therapy. Key messages Critically ill COVID-19 patients show emergency myelopoiesis. HIF1α and its transcriptionally regulated genes are expressed in immature myeloid cells which could serve as molecular targets. HIF1α and its transcriptionally regulated genes is also expressed in lung cells from critically ill COVID-19 patients which may partially explain the hypoxia related events.