Project description:Anopheline mosquitoes transmit Plasmodium parasites to humans, and are responsible for an estimated 219 million cases of malaria, leading to over 400,000 deaths annually. The mosquito’s immune system limits Plasmodium infection in several ways, and hemocytes, the insect white blood cells, are key players in these defense responses. However, the full functional diversity of mosquito hemocytes and their developmental trajectories have not been established. We use bulk RNA sequencing (scRNA-seq) to analyze the transcriptional profiles of hemocytes, of guts, and of carcasses of mosquito hemocytes in response to blood feeding or infection with Plasmodium. Data from three independent biological replicates for each condition and time-point (day 0, 1, 2, 3, and 7 after sugar-feeding, blood-feeding or P. berghei infection).
Project description:Anopheline mosquitoes transmit Plasmodium parasites to humans, and are responsible for an estimated 219 million cases of malaria, leading to over 400,000 deaths annually. The mosquito’s immune system limits Plasmodium infection in several ways, and hemocytes, the insect white blood cells, are key players in these defense responses. However, the full functional diversity of mosquito hemocytes and their developmental trajectories have not been established. We use single cell RNA sequencing (scRNA-seq) to analyze the transcriptional profiles of individual mosquito hemocytes in response to blood feeding or infection with Plasmodium. Circulating hemocytes were collected from adult A. gambiae M form (A. coluzzii) females that were either kept on a sugar meal or fed on a healthy or a Plasmodium berghei-infected mouse. Transcriptomes from 5,383 cells (collected 1, 3, and 7 days after feeding) revealed nine major cell clusters.
Project description:Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy and understanding disease progression. Redox factor-1 (Ref-1), a redox signaling protein, regulates the DNA binding activity of several transcription factors, including HIF-1. The conversion of HIF-1 from an oxidized to reduced state leads to enhancement of its DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia.Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions. Experimental Methods: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model. Matched samples were also collected for bulk proteomic analysis of the four conditions. scRNA-seq data was validated using proteomics and qRT-PCR. Ref-1’s role in mitochondrial function was confirmed using mitochondrial function assays and qRT-PCR. Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions.
Project description:Energy metabolism and extracellular matrix function are closely connected to orchestrate and maintain tissue organization, but the crosstalk is poorly understood. Here, we used scRNA-seq analysis to uncover the importance of respiration for extracellular matrix homeostasis in mature cartilage. Genetic inhibition of respiration in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage showing disorganized chondrocytes and increased deposition of extracellular matrix material. scRNA-seq analysis identified a cluster-specific decrease in mitochondrial DNA-encoded respiratory chain genes and a unique regulation of extracellular matrix-related genes in nonarticular chondrocyte clusters. These changes were associated with alterations in extracellular matrix composition, a shift in the collagen/non-collagen protein content and an increase of collagen crosslinking and ECM stiffness. The results demonstrate, based on findings of the scRNA-seq analysis, that respiration is a key factor contributing to ECM integrity and mechanostability in cartilage and presumably also in many other tissues.
Project description:Drosophila blood cells, called the hemocytes, are composed of three distinct cell populations: plasmatocytes (PM), crystal cells (CC), and lamellocytes (LM). These cell types have been extensively characterized based on the expression of a few marker genes and cell morphologies, which are inadequate to classify the complete hemocyte repertoire. Further, intermediate states along the course of maturation of these cell types have been poorly characterized. Here, we used single-cell RNA sequencing (scRNA-seq) to map the Drosophila larval hemocytes across different inflammatory conditions. We identified all known cell types, resolved PMs into different states based on the expression of genes involved in cell cycle, antimicrobial response, and metabolism together with the detection of CC and LM intermediates. Further, we discovered rare cells within CC and LM populations that express the fibroblast growth factor (FGF) ligand branchless (bnl) and receptor breathless (btl), respectively. We demonstrate that both bnl and btl are required for mediating effective immune responses against parasitoid wasp eggs in vivo, highlighting a novel role for FGF signaling in inter-hemocyte crosstalk. Our scRNA-seq analysis reveals the diversity of hemocyte populations and provides a rich resource of gene expression profiles for a systems-level understanding of their functions.
Project description:Aging is a universal biological phenomenon linked to many diseases, such as cancer or neurodegeneration. However, the molecular mechanisms underlying aging, or how lifestyle interventions such as cognitive stimulation can ameliorate this process, are yet to be clarified. Here, we performed a multi-omic profiling, including RNA-seq, ATAC-seq, ChIP-seq, EM-seq, SWATH-MS and single cell Multiome scRNA and scATAC-seq, in the dorsal hippocampus of young and old mouse subjects which were subject to cognitive stimulation using the paradigm of environmental enrichment. In this study we were able to describe the epigenomic landscape of aging and cognitive stimulation.
Project description:The Drosophila lymph gland (LG) is the larval hematopoietic organ comprised of multipotent prohemocytes and mature hemocytes and has been a valuable model for understanding mechanisms underlying the hematopoiesis and immune responses. There are three types of mature hemocytes in the lymph gland; plasmatocytes, lamellocytes, and crystal cells, all of which are analogous to myeloid-lineage blood cells. To date, the Drosophila hematopoietic system has been primarily defined by classical genetic methods that may limit the promise of its advantage as a model. In this study, we used single-cell RNA sequencing method to comprehensively profile the heterogeneity of developing myeloid hemocytes in the wild-type lymph gland and their transitions upon active immunity caused by wasp infestation. Moreover, we compared hemocytes originated from the embryonic and the lymph gland hematopoiesis and unraveled genetic and cellular diversity of two different ancestries. Finally, hemocytes of the Drosophila lymph gland and human immune cells are contrasted at a transcriptome level, highlighting evolutionary conservations of myeloid cells across species. Overall, our single-cell RNA seq analyses disclose the development of myeloid hemocytes at a single-cell level and provide comparative insights into two different lineages of hemocytes in Drosophila and perspectives on the evolution of myeloid cells, serving as an ample resource for revealing essentials of the hematopoiesis.