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[This corrects the article DOI: 10.3389/fncel.2022.1096872.].
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Signals from the microenvironment are known to be critical for development, sustaining adult stem cells, and for oncogenic progression. While candidate niche-driven signals that can promote cancer progression have been identified1-6, concerted efforts to comprehensively map microenvironmental ligands for cancer stem cell specific surface receptors have been lacking. Here, we use temporal single cell RNA-sequencing to identify molecular cues from the bone marrow stromal niche that engage leukemia stem cells (LSC) during oncogenic progression. We integrate these data with our RNA-seq analysis of human LSCs from distinct aggressive myeloid cancer subtypes and our CRISPR based in vivo LSC dependency map7 to develop a temporal receptor-ligand interactome essential for disease progression. These analyses identify the taurine transporter (TauT)-taurine axis as a critical dependency of myeloid malignancies. We show that taurine production is restricted to the osteolineage population during cancer initiation and expansion. Inhibiting taurine synthesis in osteolineage cells impairs LSC growth and survival. Our experiments with the TauT genetic loss of function murine model indicate that its loss significantly impairs the progression of aggressive myeloid leukemias in vivo by downregulating glycolysis. Further, TauT inhibition using a small molecule strongly impairs the growth and survival of patient derived myeloid leukemia cells. Finally, we show that TauT inhibition can synergize with the clinically approved oxidative phosphorylation inhibitor venetoclax8, 9 to block the growth of primary human leukemia cells. Given that aggressive myeloid leukemias continue to be refractory to current therapies and have poor prognosis, our work indicates targeting the taurine transporter may be of therapeutic significance. Collectively, our data establishes a temporal landscape of stromal signals during cancer progression and identifies taurine-taurine transporter signaling as an important new regulator of myeloid malignancies.
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While animal model studies have extensively defined the mechanisms controlling cell diversity in the developing mammalian lung, there exists a significant knowledge gap with regards to late-stage human lung development. The NHLBI Molecular Atlas of Lung Development Program (LungMAP) seeks to fill this gap by creating a structural, cellular and molecular atlas of the human and mouse lung. Transcriptomic profiling at the single-cell level created a cellular atlas of newborn human lungs. Frozen single-cell isolates obtained from two newborn human lungs from the LungMAP Human Tissue Core Biorepository, were captured, and library preparation was completed on the Chromium 10X system. Data was analyzed in Seurat, and cellular annotation was performed using the ToppGene functional analysis tool. Transcriptional interrogation of 5500 newborn human lung cells identified distinct clusters representing multiple populations of epithelial, endothelial, fibroblasts, pericytes, smooth muscle, immune cells and their gene signatures. Computational integration of data from newborn human cells and with 32,000 cells from postnatal days 1 through 10 mouse lungs generated by the LungMAP Cincinnati Research Center facilitated the identification of distinct cellular lineages among all the major cell types. Integration of the newborn human and mouse cellular transcriptomes also demonstrated cell type-specific differences in maturation states of newborn human lung cells. Specifically, newborn human lung matrix fibroblasts could be separated into those representative of younger cells (n = 393), or older cells (n = 158). Cells with each molecular profile were spatially resolved within newborn human lung tissue. This is the first comprehensive molecular map of the cellular landscape of neonatal human lung, including biomarkers for cells at distinct states of maturity.
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Perfilação da Expressão Gênica , Pulmão , Animais , Humanos , Camundongos , Pulmão/metabolismo , Mamíferos/genética , Pericitos , Fenótipo , Transcriptoma/genética , Recém-NascidoRESUMO
Postnatal lung development results in an increasingly functional organ prepared for gas exchange and pathogenic challenges. It is achieved through cellular differentiation and migration. Changes in the tissue architecture during this development process are well-documented and increasing cellular diversity associated with it are reported in recent years. Despite recent progress, transcriptomic and molecular pathways associated with human postnatal lung development are yet to be fully understood. In this study, we investigated gene expression patterns associated with healthy pediatric lung development in four major enriched cell populations (epithelial, endothelial, and nonendothelial mesenchymal cells, along with lung leukocytes) from 1-day-old to 8-yr-old organ donors with no known lung disease. For analysis, we considered the donors in four age groups [less than 30 days old neonates, 30 days to < 1 yr old infants, toddlers (1 to < 2 yr), and children 2 yr and older] and assessed differentially expressed genes (DEG). We found increasing age-associated transcriptional changes in all four major cell types in pediatric lung. Transition from neonate to infant stage showed highest number of DEG compared with the number of DEG found during infant to toddler- or toddler to older children-transitions. Profiles of differential gene expression and further pathway enrichment analyses indicate functional epithelial cell maturation and increased capability of antigen presentation and chemokine-mediated communication. Our study provides a comprehensive reference of gene expression patterns during healthy pediatric lung development that will be useful in identifying and understanding aberrant gene expression patterns associated with early life respiratory diseases.NEW & NOTEWORTHY This study presents postnatal transcriptomic changes in major cell populations in human lung, namely endothelial, epithelial, mesenchymal cells, and leukocytes. Although human postnatal lung development continues through early adulthood, our results demonstrate that greatest transcriptional changes occur in first few months of life during neonate to infant transition. These early transcriptional changes in lung parenchyma are particularly notable for functional maturation and activation of alveolar type II cell genes.
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Pulmão , Transcriptoma , Humanos , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Recém-Nascido , Lactente , Criança , Pré-Escolar , Masculino , Feminino , Análise de Sequência de RNA/métodos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Perfilação da Expressão GênicaRESUMO
The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.
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Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Humanos , Animais , Camundongos , Idoso , Medula Óssea , Células Endoteliais , Células-Tronco Hematopoéticas , Microambiente TumoralRESUMO
Glucocorticoid resistance is commonly observed in depression, and has been linked to reduced expression and/or function of the glucocorticoid receptor (NR3C1 in human, hereafter referred to as GR). Previous studies have shown that GR-mutant zebrafish exhibit behavioural abnormalities that are indicative of an affective disorder, suggesting that GR plays a role in brain function. We compared the brain methylomes and brain transcriptomes of adult wild-type and GR-mutant zebrafish, and identified 249 differentially methylated regions (DMRs) that are regulated by GR. These include a cluster of CpG sites within the first intron of fkbp5, the gene encoding the glucocorticoid-inducible heat shock protein co-chaperone Fkbp5. RNA-sequencing analysis revealed that genes associated with chaperone-mediated protein folding, the regulation of circadian rhythm and the regulation of metabolism are particularly sensitive to loss of GR function. In addition, we identified subsets of genes exhibiting GR-regulated transcription that are known to regulate behaviour, and are linked to unipolar depression and anxiety. Taken together, our results identify key biological processes and novel molecular mechanisms through which the GR is likely to mediate responses to stress in the adult zebrafish brain, and they provide further support for the zebrafish GR mutant as a model for the study of affective disorders.
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Relógios Circadianos , Receptores de Glucocorticoides , Animais , Adulto , Humanos , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Relógios Circadianos/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Encéfalo/metabolismo , Transtornos do Humor/metabolismoRESUMO
BACKGROUND: Cardiomyopathy is characterized by the pathological accumulation of resident cardiac fibroblasts that deposit ECM (extracellular matrix) and generate a fibrotic scar. However, the mechanisms that control the timing and extent of cardiac fibroblast proliferation and ECM production are not known, hampering the development of antifibrotic strategies to prevent heart failure. METHODS: We used the Tcf21 (transcription factor 21)MerCreMer mouse line for fibroblast-specific lineage tracing and p53 (tumor protein p53) gene deletion. We characterized cardiac physiology and used single-cell RNA-sequencing and in vitro studies to investigate the p53-dependent mechanisms regulating cardiac fibroblast cell cycle and fibrosis in left ventricular pressure overload induced by transaortic constriction. RESULTS: Cardiac fibroblast proliferation occurs primarily between days 7 and 14 following transaortic constriction in mice, correlating with alterations in p53-dependent gene expression. p53 deletion in fibroblasts led to a striking accumulation of Tcf21-lineage cardiac fibroblasts within the normal proliferative window and precipitated a robust fibrotic response to left ventricular pressure overload. However, excessive interstitial and perivascular fibrosis does not develop until after cardiac fibroblasts exit the cell cycle. Single-cell RNA sequencing revealed p53 null fibroblasts unexpectedly express lower levels of genes encoding important ECM proteins while they exhibit an inappropriately proliferative phenotype. in vitro studies establish a role for p53 in suppressing the proliferative fibroblast phenotype, which facilitates the expression and secretion of ECM proteins. Importantly, Cdkn2a (cyclin-dependent kinase inhibitor 2a) expression and the p16Ink4a-retinoblastoma cell cycle control pathway is induced in p53 null cardiac fibroblasts, which may eventually contribute to cell cycle exit and fulminant scar formation. CONCLUSIONS: This study reveals a mechanism regulating cardiac fibroblast accumulation and ECM secretion, orchestrated in part by p53-dependent cell cycle control that governs the timing and extent of fibrosis in left ventricular pressure overload.
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Cicatriz , Ventrículos do Coração , Camundongos , Animais , Ventrículos do Coração/patologia , Cicatriz/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Fibrose , Fibroblastos/metabolismo , Proliferação de Células , Miocárdio/metabolismoRESUMO
Mesenchymal stem/stromal cells (MSCs) within the bone marrow microenvironment (BMME) support normal hematopoietic stem and progenitor cells (HSPCs). However, the heterogeneity of human MSCs has limited the understanding of their contribution to clonal dynamics and evolution to myelodysplastic syndromes (MDS). We combined three MSC cell surface markers, CD271, VCAM-1 (Vascular Cell Adhesion Molecule-1) and CD146, to isolate distinct subsets of human MSCs from bone marrow aspirates of healthy controls (Control BM). Based on transcriptional and functional analysis, CD271+CD106+CD146+ (NGFR+/VCAM1+/MCAM+/Lin-; NVML) cells display stem cell characteristics, are compatible with murine BM-derived Leptin receptor positive MSCs and provide superior support for normal HSPCs. MSC subsets from 17 patients with MDS demonstrated shared transcriptional changes in spite of mutational heterogeneity in the MDS clones, with loss of preferential support of normal HSPCs by MDS-derived NVML cells. Our data provide a new approach to dissect microenvironment-dependent mechanisms regulating clonal dynamics and progression of MDS.
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Coronavirus disease (COVID-19) is an infectious disease caused by the SARS coronavirus 2 (SARS-CoV-2) virus. Direct assessment, detection, and quantitative analysis using high throughput methods like single-cell RNA sequencing (scRNAseq) is imperative to understanding the host response to SARS-CoV-2. One barrier to studying SARS-CoV-2 in the laboratory setting is the requirement to process virus-infected cell cultures, and potentially infectious materials derived therefrom, under Biosafety Level 3 (BSL-3) containment. However, there are only 190 BSL3 laboratory facilities registered with the U.S. Federal Select Agent Program, as of 2020, and only a subset of these are outfitted with the equipment needed to perform high-throughput molecular assays. Here, we describe a method for preparing non-hazardous RNA samples from SARS-CoV-2 infected cells, that enables scRNAseq analyses to be conducted safely in a BSL2 facility-thereby making molecular assays of SARS-CoV-2 cells accessible to a much larger community of researchers. Briefly, we infected African green monkey kidney epithelial cells (Vero-E6) with SARS-CoV-2 for 96 hours, trypsin-dissociated the cells, and inactivated them with methanol-acetone in a single-cell suspension. Fixed cells were tested for the presence of infectious SARS-CoV-2 virions using the Tissue Culture Infectious Dose Assay (TCID50), and also tested for viability using flow cytometry. We then tested the dissociation and methanol-acetone inactivation method on primary human lung epithelial cells that had been differentiated on an air-liquid interface. Finally, we performed scRNAseq quality control analysis on the resulting cell populations to evaluate the effects of our virus inactivation and sample preparation protocol on the quality of the cDNA produced. We found that methanol-acetone inactivated SARS-CoV-2, fixed the lung epithelial cells, and could be used to obtain noninfectious, high-quality cDNA libraries. This methodology makes investigating SARS-CoV-2, and related high-containment RNA viruses at a single-cell level more accessible to an expanded community of researchers.
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COVID-19 , SARS-CoV-2 , Animais , Humanos , Chlorocebus aethiops , Metanol , Acetona , Análise da Expressão Gênica de Célula Única , Células EpiteliaisRESUMO
Previous work has suggested the ability of 4-hydroxybenzoic acid (4HBA) to bind to ß -casein following ultra high temperature-like processing (UHT) in model aqueous systems. The present work confirmed directly, using MALDI-TOF-MS, the presence of covalently bound 4HBA following UHT-like treatment. In subsequent molecular dynamics simulations, the 3D structure of the ß -casein molecule was modified so that the meta-C of 4HBA ring and the side chain amino group of lys32 were linked covalently. Such simulations further indicated that the covalent addition of the phenolic compound had impacted the protein density and solvent accessibility. Hydrogen bond analysis between lys32 and the remainder of the protein structure revealed that the covalent complexation supported the formation of additional hydrogen bonds. These increased from potentially 9, in the single protein molecule, to 51 in the complex with 4HBA. However, the persistence of hydrogen bonds was reduced, leading overall to decreased stability and increased protein flexibility.
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Caseínas , Simulação de Dinâmica Molecular , Caseínas/química , Parabenos , Solventes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
COVID-19 has presented society with a public health threat greater than any in living memory, leaving us to question almost every aspect of our society. An ever increasing concern is how we protect the global population from mental illness and whether public mental health policies can achieve this. In this article I reflect on the history of mental health service development, and furthermore on how COVID-19 might impact on the delivery of public mental health strategies into the future.
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COVID-19 , Transtornos Mentais , Humanos , Saúde Mental , Saúde Pública , Pandemias , Transtornos Mentais/psicologiaRESUMO
Regular physical activity supports children's physical and psychological health and wellbeing, and provides opportunities to build social and emotional skills such as resilience, confidence, and self-efficacy. Research has demonstrated that mass participant sporting events can serve as important social and environmental correlates of physical activity. This study sought to explore parents and children's perceived motivations and perspectives of participation in the Australian Sanitarium Weet-Bix Kids TRYathlon (a non-competitive triathlon series), on children's health and well-being. An exploratory qualitative design utilizing seven focus groups were conducted with 27 family units including 31 parents and 61 children (age 7-15 years old). Data were recorded, professionally transcribed and then analyzed using thematic analysis. Three overarching themes were identified, including (1) motivations for event and physical activity participation, revealing social interaction, peer support and friendly competition as motivators for participation as well as parents' interest in supporting the development of healthy habits; (2) Perceived physical activity, fitness, and developmental benefits, detailing changes to the types of physical activity children performed as well as opportunities for children to develop physical skills and competencies; and (3) Perceived psychosocial benefits of participation, highlighting opportunities for children to develop and demonstrate independence and autonomy through event participation. Notably, parents and children identified benefits beyond immediate participation including increased family engagement and social support. Mass participant events hold the potential to elicit a range of benefits for children and their families; however, further efforts may be needed to engage less active or disengaged families.
The physical and psychological benefits of being physically active during childhood are well established. However, most Australian children do not exercise at sufficient levels to receive the full extent of these health benefits. Research has demonstrated that mass participant sporting events can create supportive environments to engage in physical activity and sport whilst promoting mental, social and emotional well-being, but their impact on youth is unknown. Therefore, this study explored parents and children's perceived motivations and perspectives of participation in a mass participant sporting event, the Australian Sanitarium Weet-Bix Kids TRYathlon, on children's health and well-being. Our research indicated a range of motivators for engaging in the event, including social interaction, peer support, friendly competition and parents' interest in supporting healthy habits. The study also highlighted numerous perceived physical and psychosocial benefits of participation, such as increased physical activity pre and post-event, improved physical competency, enhanced confidence and increased family engagement and social support. Nonetheless, we believe further efforts may be needed to engage less active or disengaged families in the Australian Sanitarium Weet-Bix Kids TRYathlon and promote behaviour change.